CN107109382A - Detergent composition, lipase Variant and the polynucleotides for encoding it - Google Patents

Detergent composition, lipase Variant and the polynucleotides for encoding it Download PDF

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CN107109382A
CN107109382A CN201580069357.8A CN201580069357A CN107109382A CN 107109382 A CN107109382 A CN 107109382A CN 201580069357 A CN201580069357 A CN 201580069357A CN 107109382 A CN107109382 A CN 107109382A
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lipase
variant
seq
acid
group
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CN107109382B (en
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K.博尔奇
J.文德
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Novo Nordisk AS
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

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  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Detergent Compositions (AREA)
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Abstract

The present invention relates to the detergent composition comprising lipase Variant.The invention further relates to the polynucleotides of these variants of lipase Variant and coding；Nucleic acid construct, carrier and host cell comprising these polynucleotides；And use the method for these variants.

Description

Detergent composition, lipase Variant and the polynucleotides for encoding it
The reference of sequence table
The application includes the sequence table of computer-reader form, is incorporated herein by reference.
Background of invention
Invention field
The present invention relates to lipase Variant, the polynucleotides of these variants are encoded, the method for these variants is produced and makes With the method for these variants.
Description of Related Art
Lipase is important biocatalyst, its shown available for it is various application and substantial amounts of different lipase It has been commercialized through identified and many.However, being suitable for making in the different components for being adapted to currently used condition New fats enzyme is desirable.
Lipase has been used in composition, so as to be made a return journey grease removal for producing aliphatic acid by hydrolyzing triglyceride Matter spot.Current detergent, cleaning and/or Fabrid care composition include many active components, these active components interference fat Fat enzyme removes the ability of lipid spot, and is in some cases unfavorable to lipase active.Lipase resistance it is such into The stability of the interference divided is desirable.In addition, such composition is not used immediately after manufacture, the result is that these fat The stability of fat enzyme may be impacted during storing.Accordingly, it would be desirable to it is active in the harsh environment of detergent composition and Stable lipase.
The invention provides the lipase Variant compared with its parent with improved characteristic.Specifically, the present invention is related to And include the variant of cysteine-cysteine (C-C) bridge.WO 99/42566 describes C-C variants, wherein addition includes half The -terminal amino acid extension of cystine, amino acid extension can be with the cysteine included in fatty enzyme amino acid sequence Form C-C bridges.
Summary of the invention
The present invention relates to the variant of parent lipase, the variant, which is included in, corresponds to SEQ ID NO:2 mature polypeptide Substitution at E1C and N233C position, with lipase active, and with SEQ ID NO:2 mature polypeptide has at least 60% but less than 100% sequence identity.
The invention further relates to encode the polynucleotides of these variants；Nucleic acid construct, carrier including these polynucleotides And host cell；And the method for producing these variants.The invention further relates to the method using these variants.
Definition
Lipase：Term " lipase (lipase) ", " lipase (lipase enzyme) ", " lipolytic enzyme ", " lipid esters Enzyme ", " steatolysis polypeptide " and " steatolysis albumen " refer to a kind of enzyme in the EC3.1.1 classes as defined in enzyme nomenclature.It can be with With lipase active (triacylglycerol lipase, EC3.1.1.3), Cutinase activity (EC3.1.1.74), sterol ester enzyme activity Property (EC3.1.1.13) and/or wax-ester hydrolase it is active (EC3.1.1.50).For purposes of the present invention, according in EXAMPLEPART Described program determines lipase active.On the one hand, variant of the invention has SEQ ID NO:The fat of 2 mature polypeptide At least the 20% of enzymatic activity, for example, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, extremely Few 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%.
Allele variant：Term " allele variant " means to take the two of a kind of gene of same chromogene seat Any one of individual or more individual alternative form.Allelic variation is naturally-produced by being mutated, and can cause many in colony State property.Gene mutation can be silence (unchanged in terms of the polypeptide of coding) or can encode with the amino acid sequence changed The polypeptide of row.The allele variant of polypeptide is by the polypeptide of the allelic variants code of gene.
cDNA：Term " cDNA " means can be by from ripe, montage the mRNA for being derived from eucaryon or prokaryotic The DNA molecular that molecule carries out reverse transcription and prepared.CDNA lacks the intron sequences that may reside in correspondence genomic DNA. Previous Initial R NA transcripts are mRNA precursors, and it will be through a series of step before the mRNA of montage of maturation is rendered as Suddenly it is processed, including montage.
Coded sequence：Term " coded sequence " means directly to indicate the polynucleotides of the amino acid sequence of variant.Code sequence The border of row is typically determined that the open reading frame is since initiation codon (such as ATG, GTG or TTG) by open reading frame And terminated with terminator codon (such as TAA, TAG or TGA).Coded sequence can be genomic DNA, cDNA, synthetic DNA or its Combination.
Control sequence：Term " control sequence " mean for expression encode the present invention variant polynucleotides necessary to Nucleotide sequence.Each control sequence can be natural (that is, from identical base for the polynucleotides for encoding the variant Cause) or external source (that is, from different genes), or be natural or external source relative to each other.These control sequences include but It is not limited to conductor, polyadenylation se-quence, propeptide sequence, promoter, signal peptide sequence and transcription terminator.At least, control Sequence processed includes promoter and transcription and translation termination signal.Be conducive to becoming these control sequences with coding for introducing The purpose of the specific restriction enzyme enzyme site of the code area connection of the polynucleotides of body, these control sequences can be provided with multiple Joint.
Expression：Term " expression " includes being related to any step of variant generation, includes but is not limited to, and is repaiied after transcription, transcription Decorations, translation, posttranslational modification and secretion.
Expression vector：Term " expression vector " means linear or ring-shaped DNA molecule, and the molecule includes the multinuclear of coding variant Control sequence that thuja acid and the polynucleotides are operationally used for its expression with offer is connected.
Fragment：Term " fragment " means to lack one or more (for example, some from the amino and/or carboxyl terminal of polypeptide It is individual) polypeptide of amino acid；Wherein the fragment has lipase active.On the one hand, maturation of the fragment comprising parent lipase is more The number of the amino acid/11 of peptide to 269 at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%th, at least 80%, at least 85%, at least 90% or at least 95%, but be less than 100%.On the one hand, the parent lipase It is SEQ ID NO:2；SEQ ID NO:4；Or SEQ ID NO:6.
High stringency conditions：Term " high stringency conditions " means for length is the probe of at least 100 nucleotides, abides by Standard DNA western blot procedure is followed, the salmon sperm sheared and be denatured in 5X SSPE, 0.3%SDS, 200 micrograms/ml at 42 DEG C Prehybridization and hybridization 12 to 24 hours in DNA and 50% formamide.2X SSC, 0.2%SDS are finally used at 65 DEG C by carrier Material is washed three times, every time 15 minutes.
Host cell：Term " host cell " means to be easy to the nucleic acid construct or table with the polynucleotides comprising the present invention Up to any cell type of carrier conversion, transfection, transduction etc..The term " host cell " covers the spawn of parental cell, The mutation occurred during due to duplication, the offspring and its parental cell are incomplete same.
Improved characteristic：Term " improved characteristic " means with the related feature being enhanced compared with parent of variant.With Parent enzyme or SEQ ID NO:2 mature polypeptide is compared, and the improved characteristic of variant of the invention is improved stability.One Aspect, the parent enzyme is SEQ ID NO:2；SEQ ID NO:4；Or SEQ ID NO:6.On the one hand, stability of the invention Can be heat endurance, the stability in the presence of proteolytic enzyme, stability in the presence of surfactants, in reducing agent In the presence of stability, stability in detergent compositions, the stability under condition of storage, under condition of storage in egg Stability, the stability under condition of storage in the presence of a reducing agent or detergent stability in the presence of white enzyme.The characteristic is led to The measure described in " example " part is crossed to determine.
Separation：Term " separation " means in non-existent form in nature or the material in environment.Separation The non-limiting examples of material include (1) any non-naturally occurring material, and (2) include but is not limited to any enzyme, variant, core Acid, albumen, any material of peptide or co-factor, the material is at least in part from one or more or all with its this qualitative correlation Removed in naturally occurring composition；(3) manually modified any material is passed through relative to the material naturally found；Or (4) pass through Relative to its natural related other components, any material for increasing the amount of the material and modifying is (for example, encode the material Multiple copies of gene；Than the use with encoding the natural stronger promoter of associated promoter of the gene of the material).Separation Material may reside in fermentation broth sample.
Low stringency condition：Term " low stringency condition " means for length is the probe of at least 100 nucleotides, abides by Standard DNA western blot procedure is followed, the salmon sperm sheared and be denatured in 5X SSPE, 0.3%SDS, 200 micrograms/ml at 42 DEG C Prehybridization and hybridization 12 to 24 hours in DNA and 25% formamide.2X SSC, 0.2%SDS are finally used at 50 DEG C by carrier Material is washed three times, every time 15 minutes.
Mature polypeptide：Term " mature polypeptide " means in translation and any posttranslational modification such as processing of N- ends, C- ends The polypeptide of its final form is in after truncation, glycosylation, phosphorylation etc..On the one hand, the mature polypeptide is SEQ ID NO:2 amino acid/11 to 269, SEQ ID NO:4 amino acid/11 is to 274 or SEQ ID NO:6 amino acid/11 is to 269.This Field is, it is known that host cell can produce the different mature polypeptides of two or more expressed by same polynucleotides (that is, has Different C- ends and/or -terminal amino acid) mixture.
Mature polypeptide encoded sequence：Term " mature polypeptide encoded sequence " means that maturation of the coding with lipase active is more The polynucleotides of peptide.On the one hand, the mature polypeptide encoded sequence is SEQ ID NO:1 nucleotides 67 to 873, SEQ ID NO:3 nucleotides 67 to 888 or SEQ ID NO:5 nucleotides 67 to 873.
Middle stringent condition：Term " middle stringent condition " means for length is the probe of at least 100 nucleotides, abides by Standard DNA western blot procedure is followed, the salmon sperm sheared and be denatured in 5X SSPE, 0.3%SDS, 200 micrograms/ml at 42 DEG C Prehybridization and hybridization 12 to 24 hours in DNA and 35% formamide.2X SSC, 0.2%SDS are finally used at 55 DEG C by carrier Material is washed three times, every time 15 minutes.
In-high stringency conditions：Term " in-high stringency conditions " means for probe that length is at least 100 nucleotides For, it then follows standard DNA western blot procedure, the salmon sheared and be denatured in 5X SSPE, 0.3%SDS, 200 micrograms/ml at 42 DEG C Prehybridization and hybridization 12 to 24 hours in fish sperm DNA and 35% formamide.2X SSC, 0.2% are finally used at 60 DEG C SDS washs carrier material three times, every time 15 minutes.
Mutant：Term " mutant " means to encode the polynucleotides of variant.
Nucleic acid construct：Term " nucleic acid construct " means list-chain or the nucleic acid molecules of double-strand, and the nucleic acid molecules are from day Separated in the gene so existed, or be modified to include the section of nucleic acid in the way of being not present in nature originally, or It is synthesis, the nucleic acid molecules include one or more control sequences.
It is operably connected：Term " being operably connected " means the coded sequence by control sequence relative to polynucleotides Placement so causes the control sequence to instruct the configuration of the expression of the coded sequence in position.
Parent or parent lipase：Term " parent " or " parent lipase " mean to be changed to produce the enzyme of the present invention The lipase of variant.The parent can be naturally occurring (wild type) polypeptide or its variant or fragment.
Reducing agent：Term " reducing agent " means to reduce, i.e. destruction is present in two in the lipase according to the present invention Any reagent or material of sulfide linkage.The example that the reducing agent of cysteine bridge (cys- bridges, C-C bridges) can be destroyed be such as, Three (2- carboxyethyls) phosphines (TCEP), dithiothreitol (DTT) (DTT), 2 mercapto ethanol (β-ME), three (3- hydroxypropyls) phosphines (THPP) and Sulphite (such as sodium sulfite).
Sequence identity：Described with parameter " sequence identity " between two amino acid sequences or two nucleotide sequences Between correlation.
For purposes of the present invention, using such as in EMBOSS bags (EMBOSS：European Molecular Biology Open software suite (The European Molecular Biology Open Software Suite), Rice (Rice) et al., 2000, heredity Trend (Trends Genet.) 16:276-277) Maimonides that (Needle) program of (preferably 5.0.0 versions or more redaction) Middle implemented Ned Coleman-wunsch (Needleman-Wunsch) algorithm (Ned Coleman (Needleman) and wunsch (Wunsch), 1970, J. Mol. BioL (J.Mol.Biol.) 48:443-453) determine between two amino acid sequences Sequence identity.Used parameter is that room starts point penalty 10, gap extension penalties 0.5 and EBLOSUM62 (BLOSUM62 EMBOSS versions) substitution matrix.To be exported labeled as the Maimonides of " most long uniformity " your (Needle) (use- Non-reduced (nobrief) option is obtained) it is used as Percent Identity and calculated as below：
(consistent residue × 100)/(comparing the room sum in length-comparison)
For purposes of the present invention, using such as in EMBOSS bags (EMBOSS：European Molecular Biology Open software suite, Rice et al., 2000, sees above) Ned Coleman implemented in the Maimonides of (preferably 5.0.0 versions or more redaction) your program- Wunsch algorithm (Ned Coleman and wunsch, 1970, see above) determines the sequence one between two deoxyribonucleotide sequences Cause property.Used parameter be Gap Opening Penalty 10, gap extension penalties 0.5, and EDNAFULL (NCBI NUC4.4's EMBOSS editions) substitution matrix.(use-non-reduced will be exported labeled as the Maimonides of " most long uniformity " your (Needle) (nobrief) option is obtained) it is used as Percent Identity and calculated as below：
(consistent deoxyribonucleotide × 100)/(comparing the room sum in length-comparison)
Subsequence：Term " subsequence " means to make one or more (for example, several) nucleotides from mature polypeptide encoded 5 ' ends of sequence and/or the polynucleotides of 3 ' end missings；Wherein subsequence coding has the fragment of lipase active.In a side Face, sub-series of packets ID containing SEQ NO:1 nucleotides 67 to 873, SEQ ID NO:3 nucleotides 67 to 888 or SEQ ID NO:The number of 5 nucleotides 67 to 873 at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%th, at least 80%, at least 85%, at least 90% or at least 95%, but be less than 100%.
Variant：Term " variant " means to include changing at one or more (for example, several) positions (that is, replace, insert Enter and/or lack) the polypeptide with lipase active.Substitution means with one position of a different aminoacids displacement occupancy Amino acid；Missing means to remove the amino acid for occupying a position；And insert and mean adjacent and follow closely and occupy a position An amino acid is added after the amino acid put.These variants of the present invention have SEQ ID NO:2 mature polypeptide is at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% lipase active.
Unusual high stringency conditions：Term " very high stringency conditions " means for spy that length is at least 100 nucleotides For pin, it then follows standard DNA western blot procedure, 5X SSPE, 0.3%SDS, 200 micrograms/ml shear and is denatured at 42 DEG C Prehybridization and hybridization 12 to 24 hours in salmon sperm dna and 50% formamide.2X SSC, 0.2% are finally used at 70 DEG C SDS washs carrier material three times, every time 15 minutes.
Unusual low stringency condition：Term " very low stringency condition " means for spy that length is at least 100 nucleotides For pin, it then follows standard DNA western blot procedure, 5X SSPE, 0.3%SDS, 200 micrograms/ml shear and is denatured at 42 DEG C Prehybridization and hybridization 12 to 24 hours in salmon sperm dna and 25% formamide.2X SSC, 0.2% are finally used at 45 DEG C SDS washs carrier material three times, every time 15 minutes.
Scourability：In the context of the present invention, removed using term " scourability " as enzyme and be present in thing to be cleaned The ability of lipid on body or the spot containing lipid.Scourability can be by calculating in the explanations of the AMSA in following methods part The so-called G/Int values of definition are quantified.Term " scourability " includes generally cleaning such as hard-surface cleaning, such as in dishwashing detergent In, but the scourability being additionally included on textile such as clothing, and also include industry cleaning and mechanism cleaning.
Wild type lipase：Term " wild type " lipase means (to be sent out such as in nature by naturally occurring microorganism Existing bacterium, yeast or filamentous fungi) expression lipase.
Variant naming rule
For purposes of the present invention, by SEQ ID NO：The mature polypeptide disclosed in 2 is to determine in another lipase Corresponding amino acid residue.By the amino acid sequence of another lipase and SEQ ID NO:The mature polypeptide disclosed in 2 It is compared, and based on the comparison, using such as in EMBOSS bags (EMBOSS：European Molecular Biology Open software suite, relies This (Rice) et al., 2000, science of heredity trend (Trends Genet.) 16:276-277) (preferably 5.0.0 editions or more redaction) Maimonides your program in implemented Ned Coleman-wunsch algorithm (Ned Coleman (Needleman) and wunsch (Wunsch), 1970, J. Mol. BioL (J.Mol.Biol.) 48:443-453) determine and SEQ ID NO:Maturation disclosed in 2 The corresponding amino acid position number of any amino acid residue in polypeptide.Used parameter is Gap Opening Penalty 10, sky Position extension point penalty 0.5, and EBLOSUM62 (BLOSUM62 EMBOSS versions) substitution matrix.
It can compare multiple peptide sequences to determine using its correspondence default parameters by using some computer programs The identification of orresponding amino acid residue in another lipase, the computer program includes but is not limited to MUSCLE (by right The expected a variety of sequences of number compare；Version 3 .5 or more redaction；Ai Dejia (Edgar), 2004, nucleic acids research (Nucleic Acids Research)32:1792-1797), MAFFT (version 6.857 or more redaction；Plus rattan (Katoh) and storehouse horse (Kuma), 2002, nucleic acids research 30:3059-3066；Plus rattan et al., 2005, nucleic acids research 33:511-518；Plus rattan and all (Toh), 2007, bioinformatics (Bioinformatics) 23:372-374；Plus rattan et al., 2009,39-64, molecular biosciences Method (Methods in Molecular Biology) 537:_39-64；Plus rattan and all, 2010,1899-1900, biology letter Breath learns 26:_ 1899-1900) and using ClustalW (1.83 or more redaction；Thomas (Thompson) et al., 1994, Nucleic acids research 22:EMBOSS EMMA 4673-4680).
When other enzymes and SEQ ID NO:2 mature polypeptide mutually away from cause traditional comparative approach based on sequence from (Linda's that (Lindahl) and Ai Luofusong (Elofsson), 2000, J. Mol. BioL when detecting its correlation (J.Mol.Biol.)295:613-615), other paired sequence comparison algorithms can be applied.In the search based on sequence more Big sensitivity can use search utility to obtain, these search utilities using the probability of peptide family represent (indicatrix) come Search for database.For example, PSI-BLAST programs produce multiple spectrograms by iterative data library searching process, and it can examine Survey remote homologue (Altschul (Atschul) et al., 1997, nucleic acids research (Nucleic Acids Res) 25:3389- 3402).If the family of polypeptide or superfamily have one or more representatives in Protein Structural Databank, it can realize very To bigger sensitivity.Program such as GenTHREADER (Jones (Jones), 1999, J. Mol. BioL (J.Mol.Biol.) 287:797-815；Mai Gufen (McGuffin) and Jones, 2003, bioinformatics (Bioinformatics) 19:874-881) Prediction is used as by the use of the information from separate sources (PSI-BLAST, secondary structure prediction, structure alignment spectrum and solvation gesture) The input for the neutral net that the structure of search sequence is folded.Similarly, high husband (Gough) et al., 2000, J. Mol. BioL (J.Mol.Biol.)313:903-919 method can be used for comparing the sequence of unknown structure and be present in SCOP databases Superfamily model.These compare and then can be used for the Homology model for producing polypeptide, and use is opened for this purpose The multiple types of tools of hair can evaluate the degree of accuracy of this class model.
For the protein of known structure, some instruments and resource can be obtained and compared for fetching with generating structure.For example, The SCOP superfamilies of albumen are compared in structure, and those comparisons are addressable and Downloadable.Can be with Using many algorithms such as apart from alignment matrix (Ao Ermu (Holm) and Sang De (Sander), 1998, protein (Proteins) 33:88-96) or combination extension (Xin Diya loves (Shindyalov) and Berne (Bourne), 1998, protein engineering (Protein Engineering)11:739-747) compare two or more protein structures, and the implementation of these algorithms Structural database of the inquiry with structures of interest can be additionally useful for, so that the structural homologue that has found that it is likely that is (for example, Ao Er Nurse and Parker (Park), 2000, bioinformatics (Bioinformatics) 16:566-567).
In the variant of the description present invention, nomenclature as described below is suitable to quote convenient.Employ accepted IUPAC Single letter and triliteral amino acid abbreviations.
Substitution.For 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, following nomenclature is used：Initial, position, substituted amino acid.Therefore, exist Threonine at position 226 is replaced by alanine to be expressed as " Thr226Ala " or " T226A ".Multiple mutation are by plus sige ("+") Separate, for example, " Gly205Arg+Ser411Phe " or " G205R+S411F " represent respectively it is sweet at position 205 and position 411 Propylhomoserin (G) is replaced by arginine (R), and serine (S) is replaced by phenylalanine (F).
Missing.For amino acid deletions, following nomenclature is used：Initial, position, *.Therefore, at position 195 Glycine deletion be expressed as " Gly195*" or " G195*”.Multiple missings are separated by plus sige ("+"), such as " Gly195*+ Ser411*" or " G195*+S411*”。
Insertion.For amino acid insertion, following nomenclature is used：Initial, position, initial, insertion ammonia Base acid.Therefore, lysine is inserted after the glycine at position 195 to be expressed as " Gly195GlyLys " or " G195GK ".Will The insertion of multiple amino acid is expressed as [initial, position, initial, insertion amino acid #1, insertion amino acid #2； Etc.].For example, inserted after the glycine at position 195 lysine and alanine be expressed as " Glyl95GlyLysAla " or “G195GKA”。
In such cases, by the way that lowercase is added to before the one or more amino acid residues inserted The one or more amino acid residues inserted are numbered in the Position Number of amino acid residue.In the above example, Therefore the sequence will be：
Parent： Variant：
195 195 195a 195b
G G-K-A
It is a variety of to change.Variant including a variety of changes is separated by plus sige ("+"), for example " Arg170Tyr+Gly195Glu " Or arginine and glycine of " R170Y+G195E " expression at position 170 and position 195 are respectively by tyrosine and glutamic acid Substitution.
Difference changes.In the case of different changes can be introduced on a position, these different changes are by comma Separate, for example " Arg170Tyr, Glu " represent that the arginine on position 170 is replaced by tyrosine or glutamic acid.Therefore, " Tyr167Gly, Ala+Arg170Gly, Ala " represent following variant：
" Tyr167Gly+Arg170Gly ", " Tyr167Gly+Arg170Ala ", " Tyr167Ala+Arg170Gly " and “Tyr167Ala+Arg170Ala”。
Detailed description of the invention
The invention provides with parent lipase or SEQ ID NO:2 mature polypeptide is compared with improved stability Lipase Variant.
Variant
The present invention relates to the variant of parent lipase, the variant, which is included in, corresponds to SEQ ID NO:2 mature polypeptide Substitution at E1C and N233C position, has at least with lipase active, and with the mature polypeptide of parent lipase 60% but less than 100% sequence identity.
On the one hand, the parent lipase is a kind of lipase, and the lipase is the polypeptide with following amino acid sequence, The amino acid sequence：(a) with the wild type fat from Humicola lanuginosa (Humicola lanuginosa) strain DSM 4109 Enzyme has at least 90% uniformity；(b) compared with the wild type lipase, it is included in E1's or Q249Interior three-dimensional knot Electroneutral or negatively charged amino acid are by the substitution of positively charged amino acid at the surface of structure；In C-terminal include peptide (c) Addition；And/or (d) meets following limitation：(i) negative electrical charge amino acid is included in the position E210 of the wild type lipase； (ii) negatively charged amino acid is included in the region of the position 90-101 corresponding to the wild type lipase；And (iii) include at the position of the N94 corresponding to the wild type lipase neutral or negative electrical charge amino acid and/or corresponding to There is negative net charge or neutral net charge in the position 90-101 of wild type lipase region.
On the one hand, the parent lipase is the lipase for having lipase active, with SEQ ID NO:2 have at least 60% But the sequence identity less than 100%, and corresponding to SEQ ID NO:2 T231R+N233R and D96E, D111A, Include taking at least one or more (for example, several) position in D254S, G163K, P256T, G91T, D27R and G38A Generation.
On the one hand, the parent lipase has SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino Acid sequence.On the one hand, the parent lipase includes SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 maturation is more Peptide is made from it.On the one hand, the variant is the variant of parent lipase, and the variant, which is included in, corresponds to SEQ ID NO:2 Mature polypeptide E1C and N233C position at substitution, with lipase active, and with SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide has at least 60% but the sequence identity less than 100%.
On the one hand, the amino acid sequence of the variant and the parent lipase have at least 60%, for example, at least 65%, extremely Few 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%th, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but the sequence identity less than 100%.
On the one hand, the variant and SEQ ID NO:2 mature polypeptide has at least 60%, for example, at least 65%, at least 70%th, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, At least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but the sequence identity less than 100%.
On the one hand, the substituted number in these variants of the invention is 1-40, such as 1-30,1-20,1- 10 and 1-5, such as 1,2,3,4,5,6,7,8,9,10,11,12,13,14, 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 Individual, 31,32,33,34,35,36,37,38,39 or 40 substitutions.
On the one hand, the variant includes SEQ ID NO:The substitution E1C+N233C of 2 mature polypeptide is made from it, or Comprising with SEQ ID NO:2 mature polypeptide have at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%th, the polypeptide with lipase active of at least 99% uniformity or be made from it, and with SEQ ID NO:2 maturation Lipase is compared, and the variant further has improved stability.On the one hand, stability of the invention can be thermally-stabilised Property, the stability in the presence of proteolytic enzyme, stability in the presence of surfactants, stabilization in the presence of a reducing agent Property, stability in detergent compositions, the stability under condition of storage, under condition of storage in the presence of protease Stability, the stability under condition of storage in the presence of a reducing agent or detergent stability.
These variants may further include in the one or more another of one or more (for example, a number) other positions Outer substitution.For example, on the one hand, the variant, which further includes to correspond to, is selected from SEQ ID NO:2 following any position One or more (for example, a number) replaces：2、4、8、11、15、27、33、38、43、48、51、54、56、57、58、60、69、 71、83、86、91、92、94、96、97、98、99、101、111、123、150、152、163、176、179、187、188、189、 198、199、200、210、216、220、224、225、227、228、229、231、236、238、239、246、249、254、255、 256、257、260、263、264、265、266、267、269.On the one hand, the variant, which is further included, corresponds to SEQ ID NO: The one or more (for example, a number) substitution of 2 any position, these substitutions are selected from：V2K、Q4R、Q4V、N8R、N11R、 Q15C、D27G、D27R、N33K、N33Q、G38A、E43C、D48C、F51V、S54T、E56K、D57G、S58A、V60K、V60S、 L69R、N71C、S83T、I86V、G91A、G91N、G91Q、N92D、N94K、N94R、D96E、D96G、D96L、D96W、L97M、 K98E、K98I、K98Q、E99K、E99N、N101D、N101S、D111A、T123V、A150G、A152G、G163K、V176L、 R179L、V187Y、V187W、Q188R、T189Y、T189W、H198S、T199R、N200R、E210K、E210Q、S216P、 Y220F、S224R、G225R、L227G、L227R、V228R、P229R、T231R、V236R、I238C、E239C、G246C、 Q249R、D254S、I255G、P256K、P256T、P256V、A257I、A257V、W260C、G263Q、L264A、I265T、 G266D、T267A、L269N、L269V.On the one hand, the variant, which is included, corresponds to SEQ ID NO:Taking at 2 following position For group or it is made from it：E1C N233C；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K N233C D254S P256T；E1C V2K D27G N33K G38A F51V D96E D111A G163K N233C D254S P256T；E1C V2K D27R N33K G38A F51V D96E D111A G163K Q188R N233C D254S P256T；E1C D27R G38A G91A N92D D96L K98Q D111A G163K N233C D254S P256T；E1C D27R G38A G91N N94R D96E D111A G163K S216P L227G N233C D254S P256T；E1C T231R N233C；E1C T231R N233C Q249R D254S；E1C G225R T231R N233C；E1C Q15C E43C T231R N233C；E1C L227R T231R N233C；E1C P229R T231R N233C；E1C L227G T231R N233C；E1C E99N N101S T231R N233C；E1C L227G T231R N233C D254S；E1C E210K L227G T231R N233C；E1C D27R N33K G38A F51V D96E K98E N101D D111A G163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V S54T E56K D57G L69R D96E K98I D111A A152G G163K T231R N233C D254S P256T；E1C V187Y T189Y L227G T231R N233C； E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C V60K I86V A150G E210K L227G T231R N233C P256K；E1C V187W T189W L227G T231R N233C；E1C N94K D96L L227G T231R N233C；E1C G91A N92D D96L K98Q L227G T231R N233C；E1C N8R L227G T231R N233C；E1C L227G V228R T231R N233C；E1C Q4R L227G T231R N233C；E1C N11R L227G T231R N233C；E1C S224R L227G T231R N233C；E1C L227G T231R N233C V236R；E1C N200R L227G T231R N233C；E1C T199R L227G T231R N233C；E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98I D111A G163K H198S Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V E56K L69R D96E K98E D111A G163K R179L T231R N233C D254S P256T A257I；E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T A257I；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T A257I；E1C D27R N33K G38A F51V S54T E56K D57G D96E K98I D111A G163K T231R N233C D254S I255G P256T A257V L269V；E1C V2K D27R N33K G38A F51V L69R D96E K98E D111A G163K V176L E210K L227G T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T； E1C D27R G38A F51V L69R D96E K98E D111A G163K E210K T231R N233C D254S P256T； And E1C N11R D27R N33K D48C F51V L69R N71C E87Q K98E N101R T143A E210K G225R L227G P229R T231R N233C Q249R P250R D254S I255G P256K。
On the one hand, except corresponding to SEQ ID NO:Outside cysteine bridge at 2 E1C N233C position, Also comprising the other cysteine bridge of one or more (for example, a number) or it is made from it according to the variant of the present invention.In a side Face, the variant includes one, two, three, four, five, six, seven, eight, nine or ten other cysteine Bridge is made from it.N- or C- ends comprising cysteine can be extended for connection to fat by such other cysteine bridge The maturing part of fat enzyme, as described in WO99/42566, and/or the cysteine bridge can connect ripe lipase Different piece, such as in naturally occurring protein.Protein natively includes cysteine bridge, and such as SEQ ID NO:2 lipase has three cysteine bridges being located at position C22-C268, C36-C41 and C104-C107.It should manage Solution, for the purpose for providing other cysteine bridge, on the one hand, can will be contained in half Guang referred in WO99/42566 The extension of propylhomoserin is added in the variant of the present invention together with replacing with E239C.Be included in the variant of the present invention other half The example of cystine bridge can be for example corresponding to SEQ ID NO:At 2 Q15C E43C and/or D48C N71C position.
These amino acid changes can have secondary properties, i.e., do not significantly affect the folding of protein and/or the guarantor of activity Keep 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor or insertion；The small missing of typically 1-30 amino acid；Small amino-or carboxyl terminal extension, such as amino Terminal Methionine residue；The small joint peptide of at most 20-25 residue；Or promote purifying by changing net charge or another function Small extension, such as polyhistidine sequence, antigenic epitopes or binding structural domain.
The example of conservative replacement is in the range of the following group：Basic amino acid (arginine, lysine and histidine), acidity Amino acid (glutamic acid and aspartic acid), polar amino acid (glutamine and asparagine), hydrophobic amino acid (leucine, Isoleucine and valine), aromatic amino acid (phenylalanine, tryptophan and tyrosine) and p1 amino acid (glycine, the third ammonia Acid, serine, threonine and methionine).The 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that specific activity will not typically be changed be it is known in the art and For example by H. Neuraths (Neurath) and R.L. Xi Er (Hill), 1979, at protein (The Proteins), science goes out Version society (Academic Press), described in New York.It is common be substituted by Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Tyr/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/ Ile, Leu/Val, Ala/Glu and Asp/Gly.
Alternately, amino acid change has a nature such that：Change the physicochemical characteristics of polypeptide.For example, amino Acid, which changes, can improve the heat endurance of polypeptide, change substrate specificity, change optimal pH etc..
The required ammonia in polypeptide can be identified according to methods known in the art, such as direct mutagenesis or alanine scanning mutagenesis Base acid (Cunningham (Cunningham) and Wei Ersi (Wells), 1989, science (Science) 244:1081-1085).Rear In a kind of technology, single alanine mutation is introduced at each residue in the molecule, and test the fat of gained mutating molecule Enzymatic activity is to identify amino acid residue that the activity to molecule is crucial.Referring further to Hilton (Hilton) et al., 1996, bioid Learn magazine (J.Biol.Chem.) 271:4699-4708.Enzyme or the active site of other biological interaction can also pass through The physical analysis of structure is determined, as being determined technology as following：Nuclear magnetic resonance, crystallography, electronic diffraction or Photoaffinity labeling, together with the mutation of the contact site amino acids to presumption.See, e.g. De Wosi (de Vos) et al., 1992, Science (Science) 255:306-312；Smith (Smith) et al., 1992, J. Mol. BioL (J.Mol.Biol.) 224:899-904；Wu Ledaweier (Wlodaver) et al., 1992, European Union of Biochemistry communication (FEBS Lett.)309:59-64.The identity of essential amino acid can also be inferred from the comparison with related polypeptide.
On the one hand, the variant have parent lipase activity at least 50%, at least 55%, at least 60%, at least 65%th, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%.
On the one hand, the variant has improved stability.
On the one hand, compared with parent enzyme, the variant has improved heat endurance.On the one hand, with parent lipase Compare, the variant is more stable at elevated temperatures.Determine to determine heat endurance by using the DSC as described in example 1.
On the one hand, compared with parent enzyme, the variant has improved stability in the presence of proteolytic enzyme.In a side Face, compared with parent lipase, the variant is more stable in the presence of proteolytic enzyme.On the one hand, compared with parent lipase, The variant in the presence of proteolytic enzyme, it is more stable at elevated temperatures.The stability improved in the presence of proteolytic enzyme It can determine to determine by using the DSC as described in example 1.
On the one hand, compared with parent enzyme, the variant has improved stability in the presence of surfactants.In a side Face, compared with parent lipase, the variant is more stable in the presence of surfactants.On the one hand, compared with parent lipase, The variant is more stable at elevated temperatures in the presence of surfactants.The stability improved in the presence of surfactants can Determined with being determined by using the DSC as described in example 1.
On the one hand, compared with parent enzyme, the variant has improved stability in the presence of a reducing agent.On the one hand, Compared with parent lipase, the variant is more stable in the presence of a reducing agent.On the one hand, compared with parent lipase, the variant In the presence of a reducing agent, it is more stable at elevated temperatures.The stability improved in the presence of a reducing agent can be by using such as NanoDSF described in example 1 determines to determine.
On the one hand, compared with parent enzyme, the variant has improved stability in detergent compositions.In a side Face, compared with parent lipase, the variant is more stable in detergent compositions.On the one hand, should compared with parent lipase Variant in detergent compositions, it is more stable at elevated temperatures.The stability improved in detergent compositions can lead to Cross and determine to determine using the DSC as described in example 1.
On the one hand, compared with parent enzyme, the variant has improved stability (i.e. with improvement under condition of storage Storage stability).On the one hand, compared with parent lipase, the variant is more stable under condition of storage.On the one hand, with parent This lipase is compared, the variant under condition of storage, it is more stable at elevated temperatures.The stability improved under condition of storage can Determined with determining option A by using the storage stability as described in example 1.
On the one hand, compared with parent enzyme, the variant has improved stabilization under condition of storage, in the presence of protease Property (i.e. with improved storage stability).On the one hand, compared with parent lipase, the variant is under condition of storage, in egg It is more stable in the presence of white enzyme.On the one hand, compared with parent lipase, the variant under condition of storage, in the presence of protease, It is more stable at elevated temperatures.The stability improved under condition of storage, in the presence of protease can be by using strictly according to the facts Storage stability described in example 1 determines option b to determine.
On the one hand, compared with parent enzyme, the variant has improved stabilization under condition of storage, in the presence of a reducing agent Property (i.e. with improved storage stability).On the one hand, compared with parent lipase, the variant is under condition of storage, also It is more stable in the presence of former agent.On the one hand, compared with parent lipase, the variant under condition of storage, in the presence of a reducing agent, It is more stable at elevated temperatures.The stability improved under condition of storage, in the presence of a reducing agent can be by using strictly according to the facts The revision that storage stability described in example 1 is determined was determined originally.
On the one hand, compared with parent enzyme, the variant has improved detergent stability.On the one hand, with parent's fat Fat enzyme is compared, and the variant is more stable in detergent.On the one hand, compared with parent lipase, the variant in detergent It is more stable at elevated temperature.The stability improved in detergent can by using as described in example 1 DSC determine or NanoDSF determines to determine.
Parent lipase
The parent lipase is：(a) a kind of polypeptide, the polypeptide and SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide has at least 60% sequence identity；(b) by a kind of polypeptide of polynucleotide encoding, the polynucleotides exist With (i) SEQ ID NO under low stringency condition:1、SEQ ID NO:3 or SEQ ID NO:5 mature polypeptide encoded sequence or (ii) the total length complement hybridization of (i)；Or (c) is by a kind of polypeptide of polynucleotide encoding, the polynucleotides and SEQ ID NO: 1、SEQ ID NO:3 or SEQ ID NO:5 mature polypeptide encoded sequence has at least 60% sequence identity.
On the one hand, the parent and SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide has At least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity, these mature polypeptides have lipase active.On the one hand, the amino acid sequence and SEQ of the parent ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide differs at most 40 amino acid, such as 1-40,1-30 Individual, 1-20,1-10,1-5, such as 1,2,3,4,5,6,7,8,9,10,11,12 It is individual, 13,14,15,16,17,18,19,20,21,22,23,24,25,26,27 It is individual, 28,29,30,31,32,33,34,35,36,37,38,39 or 40.In a side Face, the parent includes SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino acid sequence is made from it.One Aspect, the parent includes SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide is made from it.One Aspect, the parent includes SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino acid/11 is to 269 or is made from it.
On the one hand, the parent is SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 fragment, the fragment bag The NO of ID containing SEQ:2、SEQ ID NO:4 or SEQ ID NO:The number of 6 amino acid/11 to 269 at least 50%, at least 55%th, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, but it is less than 100%.On the one hand, the parent is SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 into The allele variant of ripe polypeptide.
On the one hand, the parent is by a kind of polynucleotide encoding, and the polynucleotides are in very low stringency condition, low strict bar Part, middle stringent condition, in-high stringency conditions, high stringency conditions or very under high stringency conditions with (i) SEQ ID NO:1、SEQ ID NO:3 or SEQ ID NO:5 mature polypeptide encoded sequence, or (Sa draws Brooker for the total length complement hybridization of (ii) (i) (Sambrook) et al., 1989, Molecular Cloning:A Laboratory guide (Molecular Cloning:A Laboratory Manual), The second edition, Cold SpringHarbor (Cold Spring Harbor), New York).
SEQ ID NO can be used:1、SEQ ID NO:3 or SEQ ID NO:5 polynucleotides or its subsequence, and SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 polypeptide or its fragment design nucleic acid probe so as to according to ability Known to domain method identification and clones coding from do not belong to together or species bacterial strain parent DNA.Specifically, such probe It can be used for genomic DNA or cDNA according to standard DNA western blot procedure and cell interested to hybridize, to be identified and isolated from Wherein corresponding gene.Such probe can be significantly shorter than complete sequence, but length should be at least 15, for example, at least 25, extremely Lack 35 or at least 70 nucleotides.Preferably, the nucleic acid probe length is at least 100 nucleotides, and such as length is at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, At least 700 nucleotides or at least 800 nucleotides.Two kinds of probes of DNA and RNA can be used.Probe is typically entered into rower Note (for example, with32P、3H、35S, biotin or avidin), to detect corresponding gene.The present invention covers such spy Pin.
It can screen and hybridize in the genomic DNA or cDNA library of this class other bacterial strains preparation with probe described above And encode the DNA of parent.Genomic DNA or other DNA from other such bacterial strains can pass through agarose or polyacrylamide Amine gel electrophoresis or other isolation technics are separated.DNA or the DNA of separation from library can be transferred to and be fixed on nitre On cellulose or other suitable carrier materials.In order to identify and SEQ ID NO:1 or its subsequence hybridization clone or DNA, uses carrier material in southern blotting technique.
For purposes of the present invention, hybridization shows that the polynucleotides are frequently as low as marking with one kind under very high stringency conditions non- The nucleic acid probe hybridization of note, the probe corresponds to：(i)SEQ ID NO:1、SEQ ID NO:3 or SEQ ID NO:5；(ii)SEQ ID NO:1、SEQ ID NO:3 or SEQ ID NO:5 mature polypeptide encoded sequence；(iii) its total length complement；Or (iv) its Subsequence.Such as x-ray film or any other detection means known in the art can be used to detect under these conditions The molecule of nucleic acid probe hybridization.
On the one hand, the nucleic acid probe is SEQ ID NO:1、SEQ ID NO:3 or SEQ ID NO:5 mature polypeptide is compiled Code sequence.On the one hand, the nucleic acid probe is SEQ ID NO:1、SEQ ID NO:3 or SEQ ID NO:5 nucleotides 67 to 873.On the one hand, the nucleic acid probe is a kind of polynucleotides, polynucleotide encoding SEQ ID NO:2 polypeptide；It is ripe Polypeptide；Or its fragment.On the one hand, the nucleic acid probe is SEQ ID NO:1、SEQ ID NO:3 or SEQ ID NO:5.
On the one hand, the parent is by a kind of polynucleotide encoding, the polynucleotides and SEQ ID NO:1、SEQ ID NO:3 Or SEQ ID NO:5 mature polypeptide encoded sequence have at least 60%, for example, at least 65%, at least 70%, at least 75%, extremely Few 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%th, at least 97%, at least 98%, at least 99% or 100% sequence identity.
On the one hand, the parent lipase is a kind of lipase, and the lipase is the polypeptide with following amino acid sequence, The amino acid sequence：(a) with the wild type fat from Humicola lanuginosa (Humicola lanuginosa) strain DSM 4109 Enzyme has at least 90% uniformity；(b) compared with the wild type lipase, it is included in E1's or Q249Interior three-dimensional knot Electroneutral or negatively charged amino acid are by the substitution of positively charged amino acid at the surface of structure；In C-terminal include peptide (c) Addition；And/or (d) meets following limitation：(i) negative electrical charge amino acid is included in the position E210 of the wild type lipase； (ii) negatively charged amino acid is included in the region of the position 90-101 corresponding to the wild type lipase；And (iii) include at the position of the N94 corresponding to the wild type lipase neutral or negative electrical charge amino acid and/or corresponding to There is negative net charge or neutral net charge in the position 90-101 of wild type lipase region.
On the one hand, the parent lipase is the lipase with lipase active, with SEQ ID NO:2 have at least 60% but the sequence identity less than 100%, and corresponding to SEQ ID NO:2 T231R+N233R and D96E, At at least one or more (for example, several) position in D111A, D254S, G163K, P256T, G91T, D27R and G38A Including substitution.
On the one hand, the parent is with SEQ ID NO:The polypeptide of 2 sequence.On the one hand, the parent is with SEQ ID NO:The polypeptide of 4 sequence.On the one hand, the parent is with SEQ ID NO:The polypeptide of 6 sequence.
The polypeptide can be hybrid polypeptide, and the region of one of which polypeptide is in the N- ends in the region of another polypeptide or C- Merge end.
The parent can be fused polypeptide or cleavable fused polypeptide, wherein another polypeptide is at the N- ends of polypeptide of the present invention End or the fusion of C- ends.Fusion is produced by the way that the polynucleotides for encoding another polypeptide are merged with polynucleotides of the present invention Polypeptide.Technology for producing fused polypeptide is known in the art, and including connecting the coded sequence of coded polypeptide so that They are under inframe, and control of the expression in identical promoter and terminator of fused polypeptide.In can also using The fused polypeptide of technique construction containing peptide, wherein producing fused polypeptide (cooper (Cooper) et al., 1993, European molecule upon translation Biology Society's magazine (EMBO J.) 12:2575-2583；Road gloomy (Dawson) et al., 1994, science (Science) 266: 776-779)。
Fused polypeptide may further include the cleavage site between two kinds of polypeptides.When fusion protein is secreted, the position Point is cut, so as to discharge both polypeptides.The position that the example of cleavage site is including but not limited to disclosed in the following documents Point：Martin (Martin) et al., 2003, industrial microbiology biotechnology magazine (J.Ind.Microbiol.Biotechnol.)3：568-576；Svetina et al., 2000, biotechnology magazine (J.Biotechnol.)76：245-251；Lars Ma Sen-Wilson's (Rasmussen-Wilson) et al., 1997, using with ring Border microbiology (Appl.Environ.Microbiol.) 63：3488-3493；Ward (Ward) et al., 1995, biotechnology (Biotechnology)13：498-503；And Kong Telei Lars (Contreras) et al., 1991, biotechnology 9：378- 381；Eton (Eaton) et al., 1986, biochemistry (Bi °C of hemistry) 25：505-512；Collins-Racie et al., 1995, biotechnology 13：982-987；Ka Te (Carter) et al., 1989, protein：Structure, function and science of heredity (Proteins:Structure,Function,and Genetics)6：240-248；With Glenn Stevens (Stevens), 2003, International drugs find (Drug Discovery World) 4：35-48.
The parent can obtain from the microorganism of any category.For purposes of the present invention, come as combination herein is given Term that source is used " from ... it is middle to obtain " it should mean that by the parent of polynucleotide encoding be by the source or by wherein slotting Enter the bacterial strain generation of the polynucleotides from the source.On the one hand, the parent is exocytosis.
The parent can be comprising lipase of bacterial origin.For example, the parent can be gram-positive bacterium polypeptide, such as bacillus Belong to (Bacillus), fusobacterium (Clostridium), enterococcus spp (Enter °C °C of cus), Geobacillus (Geobacillus), lactobacillus (Lactobacillus), lactococcus (Lact °C °C of cus), bacillus marinus category (° Ceanobacillus), staphylococcus (Staphyl °C °C of cus), streptococcus (Strept °C °C of cus) or streptomyces (Streptomyces) lipase；Or gramnegative bacterium polypeptide, such as campylobacter (Campylobacter), large intestine bar Bacterium (E.coli), Flavobacterium (Flavobacterium), Fusobacterium (Fusobacterium), Helicobacterium (Helicobacter), mud Bacillus (Ilyobacter), eisseria (Neisseria), pseudomonas (Pseudomonas), Salmonella (Salmonella) or Ureaplasma (Ureaplasma) lipase.
On the one hand, the parent is Alkaliphilic bacillus (Bacillus alkalophilus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus clausii), bacillus coagulans (Bacillus Coagulans), bacillus firmus (Bacillus firmus), bacillus lautus (Bacillus lautus), slow bud Spore bacillus (Bacillus lentus), bacillus licheniformis (Bacillus licheniformis), bacillus megaterium (Bacillus megaterium), bacillus pumilus (Bacillus pumilus), bacillus stearothermophilus (Bacillus stearothermophilus), bacillus subtilis (Bacillus subtilis) or the golden gemma bars of Su Yun Bacterium (Bacillus thuringiensis) lipase.
On the one hand, the parent is streptococcus equisimilis (Strept °C °C of cus equisimilis), micrococcus scarlatinae (Strept °C °C of cus pyogenes), streptococcus uberis or Malian drainage (Strept °C °C of cus equi Subsp.Zooepidemicus) lipase.
On the one hand, the parent is not streptomyces chromogenes (Streptomyces achromogenes), deinsectization streptomycete (Streptomyces avermitilis), streptomyces coelicolor (Streptomyces coelicolor), streptomyces griseus (Streptomyces griseus) or muta lead mycillin (Streptomyces lividans) lipase.
The parent can be fungal lipase.For example, the parent can be Yeast-lipase, such as candida (Candida), Kluyveromyces (Kluyveromyces), pichia (Pichia), saccharomyces (Saccharomyces), fission yeast (Schizosaccharomyces) or Ye Shi saccharomyces (Yarrowia) lipase；Or Filamentous fungi lipase, such as acremonium, Agaricus, Alternaria, aspergillus, Aureobasidium, Botryosphaeria (Botryospaeria), intend wax Pseudomonas, it is hair beak shell category, Chrysosporium, Claviceps, cochliobolus category, Coprinus, milky white Ant category, rod softgel shell category, the red shell Pseudomonas of hidden clump, Cryptococcus, Diplodia, Exidia, line black powder saccharomyces (Filibasidium), Fusarium, Gibberella, full flagellum Eimeria, Humicola, rake teeth Pseudomonas, Lentinus (Lentinula), Small chamber Coccus (Leptospaeria), Magnaporthe grisea category (Magnaporthe), black fruit Pseudomonas (Melan °C of arpus), sub- ash tree Flower Pseudomonas (Meripilus), mucor, myceliophthora, new U.S. whip Pseudomonas, Neurospora, paecilomyces, Penicillium, flat leather Pseudomonas, cud Chytridium, Poitrasia, false black Peziza, false Trichonympha, root Mucor, Schizophyllum, capital spore category, Talaromyces, thermophilic ascomycete category, the mould category of shuttle spore shell, Tolypocladium, trichoderma, Trichophaea, Verticillium, small mushroom of binding foot Category or Xylaria lipase.
On the one hand, the parent is saccharomyces carlsbergensis (Saccharomyces carlsbergensis), saccharomyces cerevisiae, saccharification Yeast, Doug Laplace yeast (Saccharomyces douglasii), Saccharomyces kluyveri (Saccharomyces Kluyveri), promise ground yeast or ellipsoideus yeast lipase.
On the one hand, the parent is solution fiber branch acremonium (Acremonium cellulolyticus), microorganism Aspergillus aculeatus (Aspergillus aculeatus), aspergillus awamori (Aspergillus awamori), smelly aspergillus (Aspergillus Foetidus), aspergillus fumigatus (Aspergillus fumigatus), aspergillus japonicus (Aspergillus japonicus), structure nest Aspergillus (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus Oryzae), the golden pityrosporion ovale (Chrysosporium inops) of straight hem, chrysosporium keratinophilum (Chrysosporium Keratinophilum), the golden pityrosporion ovale (Chrysosporium lucknowense) of clarke mire, the golden pityrosporion ovale of excrement shape (Chrysosporium merdarium), felt gold pityrosporion ovale (Chrysosporium pannicola), the golden pityrosporion ovale in Queensland (Chrysosporium queenslandicum), chrysosporium tropicum (Chrysosporium tropicum), the golden spore of band line Daughter bacteria (Chrysosporium zonatum), bar spore shape fusarium (Fusarium bactridioides), F.graminearum schw (Fusarium cerealis), storehouse prestige fusarium (Fusarium crookwellense), yellow Fusariumsp (Fusarium Culmorum), Fusarium graminearum (Fusarium graminearum), red fusarium of standing grain (Fusarium graminum), different spore sickle Spore (Fusarium heterosporum), albizzia fusarium (Fusarium negundi), Fusarium oxysporum (Fusarium Oxysporum), racemosus fusarium (Fusarium reticulatum), pink Fusariumsp (Fusarium roseum), elder Fusarium (Fusarium sambucinum), colour of skin fusarium (Fusarium sarc °C hroum), plan branch spore Fusariumsp (Fusarium sporotrichioides), sulphur color Fusariumsp (Fusarium sulphureum), circle fusarium (Fusarium Torulosum silk spore Fusariumsp (Fusarium trichothecioides), empiecement Fusariumsp (Fusarium), are intended Venenatum), grey humicola lanuginosa (Humicola grisea), Humicola insolens (Humicola insolens), Humicola lanuginosa (Humicola lanuginosa), Irpex lacteus (Irpex lacteus), rice black wool mould (Mucor miehei), thermophilic ruin Silk mould (Myceliophthora thermophila), Neuraspora crassa (Neurospora crassa), penicillium funiculosum (Penicillium funiculosum), penicillium purpurogenum (Penicillium purpurogenum), Phanerochaete chrysosporium (Phaner °C of haete chrysosporium), colourless shuttle spore shell (Thielavia achromatica), layered fusarium globosum shuttle (Thielavia albomyces), white hair shuttle spore shell (Thielavia albopilosa), Australia shuttle spore shell (Thielavia Australeinsis), excrement shuttle spore shell (Thielavia fimeti), Thielavia microspora (Thielavia microspora), ovum Spore shuttle spore shell (Thielavia ovispora), Peru's shuttle spore shell (Thielavia peruviana), hair shuttle spore shell (Thielavia setosa), knurl spore shuttle spore shell (Thielavia spededonium), heat-resisting shuttle spore shell (Thielavia Subthermophila), autochthonal shuttle spore shell (Thielavia terrestris), Trichoderma harzianum (Trichoderma Harzianum), trichodermaharzianum (Trichoderma koningii), long shoot trichoderma (Trichoderma Longibrachiatum), trichoderma reesei (Trichoderma reesei) or Trichoderma viride (Trichoderma viride) Lipase.
On the one hand, the parent is Humicola lanuginosa lipase, for example SEQ ID NO:2 lipase or its maturation are more Peptide.
It will be appreciated that for above-mentioned species, the present invention covers complete state and partial state (perfect And imperfect states) the two and other taxology equivalents, such as phorozoon, but regardless of their known things Plant title.Those of ordinary skill in the art will readily recognize the identity of appropriate equivalent.
The bacterial strain of these species can be easily for the public to obtain in many culture collections, such as U.S. typical case training Support thing collection (ATCC), Germany Microbiological Culture Collection Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ), Centraalbureau collection (Centraalbureau Voor Schimmelcultures, CBS) and american agriculture research Service Patent Culture collection northern area research Center (NRRL).
Above-mentioned probe can be used to be originated from other, including divided from nature (for example, soil, compost, water etc.) From microorganism or the DNA sample identification that is directly obtained from nature material (for example, soil, compost, water etc.) and obtain the parent. It is well known in the art for the technology directly from natural living environment separate microorganism and DNA.Then can be by another Similarly screened to obtain coding parent's in the genomic DNA or cDNA library of a kind of microorganism or hybrid dna sample Polynucleotides.Once, then can be by using to this with the polynucleotides of one or more probe in detecting to coding parent Known technology (draws Brooker to separate or clone the polynucleotides see, for example, Sa for the those of ordinary skill in field (Sambrook) et al., 1989, see above).
The preparation of variant
The invention further relates to for obtaining the method that the variant and (b) of parent lipase reclaim the variant, the variant bag It is contained in corresponding to SEQ ID NO:Substitution at the E1C and N233C of 2 mature polypeptide position, with lipase active, and With SEQ ID NO:2 mature polypeptide has at least 60% but the sequence identity less than 100%.
Any mutagenesis procedures known in the art can be used to prepare these variants, such as direct mutagenesis, synthetic gene Structure, semi-synthetic gene constructed, random mutagenesis, reorganization etc..
Direct mutagenesis is to encode introducing one or many at one or more of polynucleotides of parent restriction site The technology of individual (for example, several) mutation.
Direct mutagenesis can be realized in vitro by using the PCR for being related to the Oligonucleolide primers comprising desired mutation. Site direct mutagenesis can also be carried out by cassette mutagenesis, the cassette mutagenesis is related to by restriction enzyme including many of coding parent Cut and the oligonucleotides comprising mutation is connected in polynucleotides at site in the plasmid of nucleotides then.Generally, The restriction enzyme for digesting the plasmid and the oligonucleotides is identical, to allow the cohesive end and Insert Fragment of the plasmid each other Connection.See, e.g. thanking Le (Scherer) and Davis (Davis), 1979, (Pr ° of NAS's proceeding C.Natl.Acad.Sci.USA)76:4949-4955；With bar (Barton) et al., 1990, nucleic acids research (Nucleic Acids Res.)18:7349-4966。
Can also be by realizing direct mutagenesis in methods known in the art body.See, e.g., U.S. Patent Application Publication Number 2004/0171154；This Tosi (Storici) et al., 2001, Nature Biotechnol (Nature Biotechnol.) 19: 773-776；Kai Lun (Kren) et al., 1998, Natural medicine (Nat.Med.) 4:285-290；And Ka Lisanuo (Calissano) and graceful Cino Da Pistoia (Macino), 1996, Fungal Genetics communication (Fungal Genet.Newslett.) 43:15- 16。
Any direct mutagenesis program can be used in the present invention.In the presence of can be used for preparing many commercially available of variant Kit.
Synthetic gene, which is built, needs the polynucleotide molecule of external compounding design to encode polypeptide interested.Gene chemical synthesis It can be carried out using multiple technologies, such as by field (Tian) et al. (2004, natural (Nature) 432:Described in 1050-1054) Technology based on multichannel microchip and the similar skill that oligonucleotides is wherein synthesized and assembled on the programmable micro flow chip of light Art.
Single or multiple 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing can be made and/or insert and use mutagenesis, recombinate, and/or reorganize Known method tested, then carry out related screening sequence, such as by Reed Ha Er-Mancur Olson (Reidhaar-Olson) and Sa Aoer (Sauer), 1988, science (Science) 241:53-57；Bo Wei (Bowie) and Sa Aoer, 1989, American National section Institute's proceeding (Pr °C of .Natl.Acad.Sci.USA) 86:2152-2156；WO 95/17413；Or the disclosures of WO 95/22625 Those.Other methods that can be used include fallibility PCR, phage display (such as Lip river graceful (Lowman) et al., 1991, biology Chemistry (Bi °C of hemistry) 30:10832-10837；US5,223,409；) and regiondirected mutagenesis (Derby WO92/06204 Shi Er (Derbyshire) et al., 1986, gene (Gene) 46:145；Nellie (Ner) et al., 1988, DNA 7:127).
Mutagenesis/Shuffling Method can combine to detect the clone by host cell expression with high throughput automated screening technique Mutated polypeptides activity (Nai Si (Ness) et al., 1999, Nature Biotechnol (Nature Biotechnology) 17: 893-896).The DNA molecular of the mutagenesis of encoding active polypeptide can be reclaimed from host cell, and uses standard side in the art Method is quickly sequenced.These methods allow the rapid importance for determining single amino acids residue in polypeptide.
By the way that combinatorial compound is gene constructed, and/or direct mutagenesis, and/or random mutagenesis, and/or many aspects of reorganization It is semi-synthetic gene constructed to realize.Semi-synthetic structure typically utilizes the process combination PCR skills of the polynucleotide passage synthesized Art.Therefore, the region of the restriction of gene can be with de novo formation, and other regions can use site-specific mutagenesis primer to expand Increase, and also have other regions to be subjected to fallibility PCR or non-fallibilities PCR amplifications.Then polynucleotides subsequence can be carried out Reorganization.
Polynucleotides
The invention further relates to the polynucleotides for the variant for encoding the present invention.
Nucleic acid construct
The invention further relates to including coding the present invention it is variant, be operably coupled in one or more control sequences Polynucleotides nucleic acid construct, one or more control sequences instruct code sequence under conditions of compatible with control sequence It is listed in the expression in suitable host cell.
The polynucleotides can be manipulated in many ways to provide the expression of variant.Depending on expression vector, in multinuclear It can be desirable or required to carry out manipulation before thuja acid insertion vector to it.For utilizing recombinant DNA method modification The technology of polynucleotides is well known in the art.
Control sequence can be promoter, i.e., recognize the polynucleotides for expressing the polynucleotides by host cell.Open Mover includes the transcriptional control sequence for the expression for mediating the variant.Promoter can be that transcriptional activity is shown in host cell Any polynucleotides, including saltant type, truncated-type and hybrid promoters, and can be same with the host cell by encoding Source or heterologous extracellular or intracellular polypeptides gene are obtained.
The example of suitable promoter for the transcription for the nucleic acid construct that the present invention is instructed in bacterial host cell is The promoter obtained from following gene：Bacillus amyloliquefaciens alpha-amylase gene (amyQ), bacillus licheniformis alpha-starch Enzyme gene (amyL), bacillus licheniformis penicillinase gene (penP), bacillus stearothermophilus production maltogenic amylase base Because of (amyM), subtilis levansucrase gene (sacB), bacillus subtilis xylA and xylB gene, Su Yunjin Bacillus cryIIIA genes (A Gaisai (Agaisse) and Le Erkelv (Lereclus), 1994, molecular microbiology (Molecular Microbiology)13:97-107), E. coli lac operon, Escherichia coli trc promoters (Ai Gong (Egon) et al., 1988, gene (Gene) 69:301-315), streptomyces coelicolor agarase gene (dagA), Yi Jiyuan Core beta-lactam enzyme gene (Wella-Karma love (Villa-Kamaroff) et al., 1978, NAS's proceeding (Pr°C.Natl.Acad.Sci.USA)75:3727-3731) and tac promoters (moral bohr (DeBoer) et al., 1983, it is beautiful State's Proceedings of the National Academy of Sciences 80:21-25).Other promoters are described in gilbert (Gilbert) et al., 1980, the science U.S. People (Scientific American) 242:74-94 " useful proteins matter (the Useful proteins from recombinant bacteria from recombinant bacteria)”；And in Pehanorm Brooker (Sambrook) et al., 1989, see above.Series connection is opened The example of mover is disclosed in WO 99/43835.
For the reality for the suitable promoter for instructing transcription of the nucleic acid construct of the present invention in filamentous fungal host cell Example is the promoter obtained from the gene of the following：Aspergillus nidulans acetamidase, Aspergillus ni ger neutral alpha-amylase, aspergillus niger acid Stability alpha-amylase, aspergillus niger or Aspergillus awamori amylase (glaA), oryzae TAKA amylase, Aspergillus oryzae alkaline egg White enzyme, aspergillus oryzae triose-phosphate isomerase, sharp fusarium trypsin like proteases (WO96/00787), empiecement fusarium starch glucose Glycosides enzyme (WO 00/56900), empiecement fusarium Daria (Fusarium venenatum Daria) (WO 00/56900), empiecement sickle Spore Quinn (Fusarium venenatum Quinn) (WO00/56900), rhizomucor miehei (Rhizomucor miehei) fat Fat enzyme, rhizomucor miehei aspartic protease, trichoderma reesei β-glucosyl enzym, trichoderma reesei cellobiohydrolase I, Richter scale Trichoderma cellobiohydrolase II, trichoderma reesei endoglucanase I, trichoderma reesei endoglucanase II, in trichoderma reesei Cut dextranase III, trichoderma reesei endoglucanase IV, trichoderma reesei endoglucanase V, Xylanase from Trichoderma reesei I, Xylanase from Trichoderma reesei II, trichoderma reesei xylobiase, and (a kind of promoter of modification, it comes NA2-pi promoters From aspergillus neutral alpha-amylase enzyme gene, wherein untranslated conductor is by the untranslated of aspergillus triose phosphate isomerase gene Conductor substitute；Non-limiting examples include the promoter of the modification of the aspergillus niger gene from encoding neutral alpha-amylase, Aspergillus nidulans or the untranslated conductor of aspergillus oryzae gene wherein for own coding triose-phosphate isomerase is replaced not The conductor of translation)；And its saltant type, truncated-type and hybrid promoters.
In yeast host, useful promoter is obtained from the gene of the following：Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose-phosphate isomerase (TPI), brewing yeast metallothionein (CUP1) and saccharomyces cerevisiae 3-phoshoglyceric acid Kinases.Rome Northey (Romanos) et al., 1992, yeast (Yeast) 8:423-488 describes other of yeast host cell Useful promoter.
Control sequence can also be to be recognized to terminate a kind of transcription terminator of transcription by host cell.The terminator sequence It is operably connected to 3 '-end of the polynucleotides for encoding the variant.It can use and have functional times in host cell What terminator.
The preferred terminator of bacterial host cell is obtained from the gene of the following：Bacillus clausii alkali protease (aprH), bacillus licheniformis alpha-amylase (amyL) and Escherichia coli rRNA (rrnB).
The preferred terminator of filamentous fungal host cell is obtained from the gene of the following：Aspergillus nidulans neighbour's amino Benzoic acid synthase, aspergillus niger glucoamylase, aspergillus niger alpha-Glucosidase, oryzae TAKA amylase and sharp fusarium tryptose Enzyme sample protease.
Preferred terminator for yeast host cell is obtained from the gene of the following：Saccharomyces cerevisiae enolase, wine brewing Yeast cells pigment C (CYC1) and S. cerevisiae glyceraldehyde -3- phosphate dehydrogenases.Northey (Romanos) et al. in Rome Other useful terminators of yeast host cell are described in (1992, see above).
Control sequence can also be the stable sub-districts of the mRNA of the upstream of coding sequence of promoter downstream and gene, and it increases should The expression of gene.
The example of the stable sub-district of suitable mRNA is from bacillus thuringiensis cryIIIA genes (WO 94/25612) and withered Careless bacillus SP82 genes, which are obtained, (stops (Hue) et al., 1995, Bacteriology (Journal of Bacteriology) 177:3465-3471)。
The control sequence can also be conductor, and the conductor is the mRNA important to host cell translation untranslated Area.Leader sequence is operably coupled to 5 '-end of the polynucleotides for encoding the variant.What is worked in host cell appoints What conductor can be used.
Preferred conductor for filamentous fungal host cell is from oryzae TAKA amylase and aspergillus nidulans triose phosphorus The gene of acid isomer enzyme is obtained.
Conductor suitable for yeast host cell is obtained from the gene of the following：Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae glycerol 3-phosphate acid kinase, cerevisiae alpha-factor and saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenation Enzyme (ADH2/GAP).
The control sequence can also be Polyadenylation sequences, that is, be operably connected to the variant coding sequences 3 '-end and the signal that is identified as being added to polyadenosine residues on transcribed mRNA by host cell when transcription Sequence.Any Polyadenylation sequences worked in host cell can be used.
Preferred polyadenylation se-quence for filamentous fungal host cell is obtained from the gene of the following：Structure nest is bent Mould anthranilate synthase, aspergillus niger glucoamylase, aspergillus niger alpha-Glucosidase, oryzae TAKA amylase and sharp sickle Spore trypsin like proteases.
For the useful polyadenylation se-quence of yeast host cell is in Guo (Guo) and thanks to Germania (Sherman), 1995, Molecular cytobiology (Mol.Cellular Biol.) 15:Described in 5983-5990.
The control sequence can also be signal peptide coding region, encode the signal peptide being connected with the N- ends of variant, and guide The variant enters the secretion path of cell.The 5 ' of the coded sequence of the polynucleotides-end inherently can be encoded comprising signal peptide Sequence, the section of coded sequence of the signal coding sequence in translation reading frame with encoding the variant is natively connected to one Rise.Alternately, coded sequence 5 '-end can include the signal coding sequence for for the coded sequence being external source.In coding In the case that sequence does not natively include signal coding sequence, it may be necessary to foreign signal peptide coding sequence.Alternately, outside Source signal peptide-coding sequence can be with substitute simply natural signals peptide-coding sequence, to strengthen the secretion of variant.However, it is possible to The variant of instruction expression enters any signal coding sequence of the secretion path of host cell.
Useful signal peptide-coding sequence for bacterial host cell is formed sediment from the Fructus Hordei Germinatus of bacillus NCIB 11837 sugar Powder enzyme, bacillus licheniformis subtilopeptidase A, Di clothing Ya spore Gan Jun Calcium-lactamase, Shi heat fat Ya spore Gan Jun Ru-shallow lake What the gene of powder enzyme, stearothermophilus neutral protease (nprT, nprS, nprM) and bacillus subtilis prsA was obtained Signal coding sequence.Other signal peptide is by Xi Moning (Simonen) and Paar watt (Palva), 1993, microorganism comment (Microbiological Reviews)57:109-137 is described.
Useful signal peptide-coding sequence for filamentous fungal host cell is from Aspergillus ni ger neutral amylase, aspergillus niger Portugal Saccharogenic amylase, oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens EGV, pubescence detritus The signal coding sequence that the gene of mould lipase and rhizomucor miehei aspartic protease is obtained.
The useful signal for yeast host cell is obtained from the gene of cerevisiae alpha-factor and Saccharomyces cerevisiae invertase Peptide.Other useful signal coding sequences are described by Romano this (Romanos) et al. (1992, see above).
The control sequence can also be propeptide code sequence of the coding positioned at the propetide of the N- ends of variant.The polypeptide of generation It is referred to as preemzyme (proenzyme) or propolypeptide (or being referred to as proenzyme (zymogen) in some cases).Propolypeptide is usual It is inactive and active peptides from the propetide of propolypeptide can be converted into by catalysis cutting or autocatalysis cutting. Can be from bacillus subtilis alkali proteinase (aprE), Bacillus subtilis neutral protease (nprT), thermophilic fungus destroyed wire paint The gene of enzyme (WO 95/33836), rhizomucor miehei aspartic protease and Niang wine Jiao Mu Ru-factor obtains propetide code sequence Row.
In the presence of signal peptide sequence and propeptide sequence, the propeptide sequence is located immediately adjacent the variant N- ends and the signal peptide sequence are located immediately adjacent the N- ends of the propeptide sequence.
Also desirable can be the regulation sequence that grows expression to adjust the variant of the addition relative to host cell Row.The example of regulating system is in response to cause those that the expression of gene is turned on and off in chemical or physical stimulus, including The presence of regulating compound.Regulating system in prokaryotic system includes lac, tac and trp operon system.In yeast, ADH2 systems or GAL1 systems can be used.In filamentous fungi, aspergillus niger glucose starch enzyme promoters, aspergillus oryzae can be used TAKA alpha-amylases promoter and aspergillus oryzae glucose starch enzyme promoters.Other examples of regulatory sequence are that those allow base The sequence of gene-amplification.In eukaryotic system, these regulating and controlling sequences are included in the dihydrofoilic acid being amplified in the presence of methotrexate (MTX) also Nitroreductase gene and the metallothionein gene expanded with heavy metal.In these cases, the polynucleotides for encoding the variant will It is operably connected with the regulatory sequence.
Expression vector
Terminated the invention further relates to polynucleotides, promoter and the transcription and translation of the variant including the coding present invention The recombinant expression carrier of signal.Different nucleotides and control sequence can link together to produce recombinant expression carrier, this One recombinant expression carrier can include one or more easily restriction sites to allow to insert or take at these sites In generation, encodes the polynucleotides of the variant.Alternately, can be by the nucleic acid construct by polynucleotides or comprising the polynucleotides Body inserts in the suitable carrier for expression and expresses the polynucleotides.When producing the expression vector, the coded sequence is located at In the carrier, so that the coded sequence is operably connected with the suitable control sequence that the confession is expressed.
Recombinant expression carrier can be can easily carry out recombinant DNA program and can cause polynucleotides express it is any Carrier (for example, plasmid or virus).The selection of carrier will typically depend on the host cell of the carrier and the carrier to be introduced Compatibility.The carrier can be linear or closure cyclic plasmid.
The carrier can be autonomously replicationg vector, i.e. the carrier existed as extrachromosomal entity, and it is replicated independently of dye Colour solid is replicated, for example, plasmid, extra-chromosomal element, minichromosomes or artificial chromosome.The carrier can include any to ensure The key element of self-replacation.Alternately, the carrier can be such carrier, when it is introduced into the host cell, be integrated Replicated into genome and together with wherein having incorporated its one or more chromosomes.In addition it is possible to use single carry (these carriers or plasmid jointly comprise the gene to be introduced into host cell for body or plasmid or two or more carriers or plasmid STb gene in group) or transposons.
The carrier, which is preferably comprised, one or more allows easily to select transformed cells, transfectional cell, transducer cell etc. thin The selected marker of born of the same parents.Selected marker is such a gene, and the product of the gene provides biocide resistance or virus Resistance, heavy metal resistance, auxotrophic prototrophy etc..
The example of bacillary selected marker is bacillus licheniformis or bacillus subtilis dal genes, or assigns antibiosis The mark of plain resistance (such as ampicillin, chloramphenicol, kanamycins, neomycin, spectinomycin or tetracyclin resistance).For The suitable mark of yeast host cell includes but is not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selected marker for being used in filamentous fungal host cell includes but is not limited to amdS (acetamidase), argB (bird ammonia Sour carbamylrtansferase), bar (phosphine oxamate transacetylase), hph (hygromix phosphotransferase), niaD (nitrate reductases Enzyme), pyrG (orotidine -5 '-phosphate decarboxylase), (ortho-aminobenzoic acid is closed by sC (sulfate adenylyl transferase) and trpC Enzyme), together with its equivalent.It is preferred that used in Aspergillus cell aspergillus nidulans or aspergillus oryzae amdS and pyrG gene and Streptomyces hygroscopicus bar genes.
Carrier preferably comprise permission vector integration into the genome of host cell or carrier in cell independently of gene One or more elements of group autonomous replication.
For being incorporated into the host cell gene group, the carrier can by encode the variant polynucleotide sequence or For any other element by homologous or non-homologous re-combination to the carrier in the genome.Alternately, the load Body, which can be included, to be used to instruct to be incorporated into by homologous recombination in one or more of host cell gene group chromosome The other polynucleotides of one or more exact positions.In order to increase the possibility integrated in exact position, what these were integrated Element should include sufficient amount of nucleic acid, such as 100 to 10,000 base-pair, 400 to 10,000 base-pair and 800 To 10,000 base-pairs, these base-pairs with corresponding target sequence there is the sequence identity of height to improve homologous recombination Possibility.These integrated elements can be the homologous any sequence of target sequence in the genome with host cell.In addition, these Integrated element can be non-coding polynucleotide or coded polynucleotide.On the other hand, the carrier can be by non-homogeneous heavy Group is incorporated into the genome of host cell.
For autonomous replication, the carrier, which may further include, enables the carrier autonomous in the host cell discussed The replication orgin of duplication.Replication orgin can be any plasmid replicon of the mediation autonomous replication worked in cell.Art Language " replication orgin " or " plasmid replicon " mean the polynucleotides for enabling plasmid or carrier to replicate in vivo.
The example of bacterial origin of replication be allow replicated in Escherichia coli pBR322 plasmid, pUC19, pACYC177, And pACYC184 replication orgin, and allow replicated in bacillus plasmid pUB110, pE194, pTA1060, And pAM β 1 replication orgin.
Example for the replication orgin in yeast host cell be 2 micron origin of replication, ARS1, ARS4, ARS1 and CEN3 combination and ARS4 and CEN6 combination.
In filamentous fungal cells the example of useful replication orgin be AMA1 and ANS1 (Ge Musi (Gems) et al., 1991, gene (Gene) 98:61-67；Card human relations (Cullen) et al., 1987, nucleic acids research (Nucleic Acids Res.) 15: 9163-9175；WO 00/24883).Separation and the bag of AMA1 genes can be completed according to the method disclosed in WO 00/24883 The structure of plasmid or carrier containing the gene.
The polynucleotides Insertion Into Host Cell of the invention of more than one copy can be increased the generation of variant.Pass through At least one other copy of sequence is incorporated into host cell gene group or by including one and the polynucleotides one The amplifiable selected marker risen can obtain the increased copy number of polynucleotides, wherein by appropriate choosing Cell is cultivated in the presence of selecting property reagent can select cell, the Yi Jiyou of the copy through amplification comprising selected marker The other copy of this polynucleotides.
With the program of recombinant expression carrier for building the present invention it is the common of this area for connecting above-described element Known to technical staff (see, e.g., Pehanorm Brooker (Sambrook) et al., 1989, see above).
Host cell
The invention further relates to recombinant host cell, these recombinant host cells include coding the present invention it is variant, can grasp It is connected to the polynucleotides of one or more control sequences with making, one or more control sequences instruct the variant of the present invention Produce.Construct or carrier including polynucleotides are introduced into host cell, so that the construct or carrier are maintained As chromosomal integrant or as the external carrier of dyeing of autonomous replication, as noted earlier.Term " host cell " cover by The spawn of the mutation that occurs in the reproduction process parental cell different from parental cell.The selection of host cell is very big By depending on the gene and its source for encoding the variant in degree.
Host cell can be useful any cell in restructuring produces variant, such as prokaryotic or eukaryotic.
Prokaryotic host cell can be any Gram-positive or gramnegative bacterium.Gram-positive bacterium include but It is not limited to：Bacillus, fusobacterium, enterococcus spp, Geobacillus, lactobacillus, lactococcus, bacillus marinus Category, staphylococcus, streptococcus and streptomyces.Gramnegative bacterium includes but is not limited to：It is campylobacter, big Enterobacteria, Flavobacterium, Fusobacterium, Helicobacterium, mud Bacillus, eisseria, pseudomonas, Salmonella, And Ureaplasma.
Bacterial host cell can be any bacillus cell, include but is not limited to：Alkaliphilic bacillus, solution starch It is bacillus, bacillus brevis, Bacillus circulans, Bacillus clausii, bacillus coagulans, bacillus firmus, bright Rotten bacillus, bacillus lentus, bacillus licheniformis, bacillus megaterium, bacillus pumilus, stearothermophilus gemma bar Bacterium, bacillus subtilis and Bacillus thuringiensis cell.
Bacterial host cell can also be any streptococcus cell, include but is not limited to：Streptococcus equisimilis, suppurative chain Coccus, streptococcus uberis and Malian drainage cell.
Bacterial host cell can also be any Streptomyces cell, include but is not limited to：Not streptomyces chromogenes, deinsectization chain Mould, streptomyces coelicolor, streptomyces griseus and muta lead mycillin cell.
DNA, which is introduced into bacillus cell, to be realized by following：Protoplast transformation (see, for example, (Chang) and Koln (Cohen), 1979, molecular genetics and genomics (Mol.Gen.Genet.) 168:111-115), feel By state cell transformation (see, e.g., poplar lattice (Young) and Spizien (Spizizen), 1961, Bacteriology (J.Bacteriol.)81:823-829；Or Du Bainu (Dubnau) and David Du Fu-Abbe Ademilson (Davidoff- Abelson), 1971, J. Mol. BioL (J.Mol.Biol.) 56:209-221), electroporation is (see, e.g., Mao Chuan (Shigekawa) and dongle (Dower), 1988, biotechnology (Biotechniques) 6:742-751) or engagement (ginseng See, for example gram Le (Koehler) and Sohne (Thorne), 1987, Bacteriology 169:5271-5278).DNA is introduced big It can be realized in coli cell by following：Protoplast transformation is (see, e.g., Hana sweat (Hanahan), 1983, molecule Biology magazine (J.Mol.Biol.) 166:557-580) or electroporation is (see, e.g., dongle (Dower) et al., 1988, core Acid research (Nucleic Acids Res.) 16:6127-6145).DNA is introduced into Streptomyces cell can be by following come real It is existing：Protoplast transformation, electroporation are (see, for example, tribute (Gong) et al., 2004, microbiology grand sight (Folia Microbiol.) (Prague (Praha)) 49:399-405), engage (see, for example, Ma Zuodiye (Mazodier) et al., 1989, Bacteriology (J.Bacteriol.) 171:3583-3585) or transduction (see, for example, primary gram (Burke) et al., 2001, NAS's proceeding (Pr °C of .Natl.Acad.Sci.USA) 98:6289-6294).DNA is introduced into false monospore It can be realized in Pseudomonas cell by following：Electroporation (see, e.g., Cai (Choi) et al., 2006, micro-biological process is miscellaneous Will (J.Microbiol.Methods) 64：391-397) or engage (see, e.g., intracutaneous many (Pinedo) and Si Meici (Smets), 2005, using with environmental microbiology (Appl.Environ.Microbiol.) 71:51-57).DNA is introduced into chain It can be realized in Coccus cell by following：Natural competence is (see, e.g., Perry (Perry) He Zangman (Kuramitsu), 1981, infection is with being immunized (Infect.Immun.) 32:1295-1297), protoplast transformation is (referring to example Such as, Ka Te (Catt) and Zhuo Linke (Jollick), 1991, microorganism (Microbios) 68:189-207), electroporation (referring to, For example, Bark profit (Buckley) et al., 1999, using with environmental microbiology 65:3800-3804) or engage (referring to example Such as, gram sharp Weir (Clewell), 1981, Microbi (Microbiol.Rev.) 45:409-436).However, it is possible to make With any method known in the art that DNA is introduced to host cell.
Host cell can also be eucaryote, such as mammal, insect, plant or fungal cell.
Host cell can be fungal cell." fungi " includes Ascomycota (Ascomycota), load as used herein Daughter bacteria door (Basidiomycota), chytridiomycota (Chytridiomycota) and Zygomycota (Zygomycota) and oomycota (Oomycota) and all mitosporic fungis (as defined by Hawkesworth (Hawksworth) et al., quoted from： Pacify Ellsworth (Ainsworth) and than this fungi dictionary (Dictionary of The Fungi) than (Bisby), the 8th Version, 1995, international CAB, university press (University Press), Cambridge, Britain).
Fungal host cells can be yeast cells." yeast " includes ascosporogenous yeast as used in this (ascosporogenous yeast) (Endomycetale (Endomycetales)), basidiosporogenous yeast (basidiosporogenous yeast) and belong to Fungi Imperfecti (Fungi Imperfecti) (gemma guiding principle (Blastomycetes) yeast).Because the classification of yeast may change in future, for purposes of the present invention, yeast should (Skinner (Skinner), Paasche are not with active (Biology and Activities of Yeast) for such as biology of yeast That (Passmore) and Davenport (Davenport) write, (S ° of Applied Bacteriology Society's symposium series 9 C.App.Bacteriol.Symposium Series No.9), 1980) described by define like that.
Yeast host cell can be Candida cell, Hansenula cells, Kluyveromyces cell, Bi Chi Saccharomyces cell, Blastocystis cell, fission yeast or Ye Luoweiya Saccharomyces cells, such as Kluyveromyces lactis cell, card Family name's yeast cells, brewing yeast cell, saccharomyces diastaticus cell, Douglas yeast (Saccharomyces douglasii) are thin Born of the same parents, Saccharomyces kluyveri cell, promise ground yeast cells, oviformis cell or Yarrowialipolytica cell.
The fungal host cells can be filamentous fungal cells." filamentous fungi " includes Eumycota (Eumycota) and oomycetes All filamentous forms (such as by defined in Hawkesworth et al. (1995, see above)) of door (Oomycota) subclass.Generally, Filamentous fungi is characterised by being made up of chitin, cellulose, glucan, chitosan, mannosan and other complicated polysaccharide Mycelia body wall.Nutrient growth is extended by mycelia, and carbon catabolism is obligate aerobic.On the contrary, yeast is (such as wine brewing ferment It is female) nutrient growth be budding (budding) by unicellular thallus, and carbon catabolism can be fermentable.
Filamentous fungal host cell can be Acremonium, aspergillus, the mould category (Bjerkandera) of Aureobasidium, smoke pipe, Intend cured Pseudomonas, Chrysosporium, Coprinus, Coriolus Qu61 (Coriolus), Cryptococcus, Filobasidiaceae (Filibasidium), Fusarium, Humicola, Magnaporthe grisea category, mucor, myceliophthora, new U.S. whip Pseudomonas, Neurospora, Paecilomyces, Penicillium, flat lead fungi category, penetrate arteries and veins Pseudomonas (Phlebia), cud Chytridium, Pleurotus (Pleurotus), split pleat Pseudomonas, Talaromyces, thermophilic ascomycete category, Thielavia, Tolypocladium, Trametes (Trametes) or trichoderma cell.
For example, filamentous fungal host cell can be aspergillus awamori, smelly aspergillus, aspergillus fumigatus, aspergillus japonicus, aspergillus nidulans, Aspergillus niger, aspergillus oryzae, black thorn smoke pipe bacterium (Bjerkandera adusta), dry plan wax bacterium (Ceriporiopsis Aneirina), Ka Neiji intends wax bacterium (Ceriporiopsis caregiea), pale yellow plan wax pore fungi (Ceriporiopsis Gilvescens), Pernod wishes tower plan wax bacterium (Ceriporiopsis pann °C inta), annulus plan wax bacterium (Ceriporiopsis Rivulosa), micro- red plan wax bacterium (Ceriporiopsis subrufa), worm intend wax bacterium (Ceriporiopsis Subvermispora), the golden pityrosporion ovale (Chrysosporium inops) of straight hem, chrysosporium keratinophilum, Lu Kenuo trains of thought gold The golden pityrosporion ovale (Chrysosporium merdarium) of pityrosporion ovale (Chrysosporium lucknowense), excrement shape, rent Pityrosporion ovale, the golden pityrosporion ovale (Chrysosporium queenslandicum) in Queensland, chrysosporium tropicum, brown thin golden pityrosporion ovale (Chrysosporium zonatum), Coprinus cinereus (Coprinus cinereus), hairy fungus (Coriolus Hirsutus), bar spore shape fusarium, cereal fusarium, storehouse prestige fusarium, machete fusarium, F.graminearum schw, red fusarium of standing grain, different spore fusarium, conjunction Joyous wood fusarium, sharp fusarium, racemosus fusarium, pink fusarium, elder fusarium, colour of skin fusarium, intend branch spore fusarium, sulphur color fusarium, Circle fusarium, plan silk spore fusarium, empiecement fusarium, Humicola insolens, Humicola lanuginosa, rice black wool mould, thermophilic fungus destroyed wire, coarse chain spore Bacterium, penicillium purpurogenum, the yellow flat lead fungi of spore (Phaner °C of haete chrysosporium), penetrate arteries and veins bacterium (Phlebia radiata), Pleurotus eryngii (Pleurotus eryngii), autochthonal shuttle spore shell are mould, long domain Trametes trogii (Trametes villosa), discoloration bolt Bacterium (Trametes versicolor), Trichoderma harzianum, trichodermaharzianum, long shoot trichoderma, trichoderma reesei or Trichoderma viride cell.
Fungal cell can be in a way known by be related to protoplast formation, the conversion of protoplast and The process of the regeneration of cell membrane is converted.For converting the suitable program description of aspergillus and pyr-trichoderma host cell in EP 238023；Yue Erdun (Yelton) et al., 1984, NAS's proceeding (Pr °C of .Natl.Acad.Sci.USA) 81: 1470-1474；With Harald Christensen (Christensen) et al., 1988, biotechnology (Bio/Technology) 6:1419- In 1422.For converting the appropriate methodology of Fusarium species by horse traction Deere (Malardier) et al., 1989, gene (Gene)78:147-156 and WO 96/00787 are described.It can use by the program transformed yeast as described in documents below： Bake that (Becker) and melon human relations are special (Guarente), at Abbe Ademilson (Abelson), J.N. and simon (Simon), M.I. Compile, yeast geneticses and Molecular Biology (Guide to Yeast Genetics and Molecular Biology), Enzymology method (Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology), volume 194, the 182-187 pages, Co., Ltd of academic press (Academic Press, Inc.), New York； Her rattan (Ito) et al., 1983, Bacteriology (J.Bacteriol.) 153:163；And Heng Neien (Hinnen) et al., 1978, NAS's proceeding (Pr °C of .Natl.Acad.Sci.USA) 75:1920.
The method of generation
The invention further relates to produce the method for variant, these methods include：(a) suitable for expressing training under conditions of the variant Support the host cell of the present invention；And reclaim the variant (b).
These host cells are trained using methods known in the art in the nutrient medium for being suitable for producing variant Support.For example, can be by Shaking culture, or in suitable culture medium and allowing the bar of variant expression and/or separation Carried out under part in laboratory or industrial fermentation tank small-scale or large scale fermentation (including continuously ferment, batch fermentation, in batches to Material fermentation or solid state fermentation) cultivate the cell.The culture is to use program as known in the art, is trained in a kind of suitable nutrition Support in base and occur, the culture medium includes carbon and nitrogen source and inorganic salts.Suitable culture medium can obtain from commercial supplier or can To be prepared according to disclosed composition (for example, in catalogue of American type culture collection).If the variant is secreted Into the nutrient medium, then the variant can be reclaimed directly from the culture medium.If the variant is not secreted, it can be from thin Reclaimed in cellular lysate liquid.
The method special to these variants known in the art can be used to detect these variants.These detection methods include But it is not limited to, the use of specific antibody, the formation of enzyme product or the disappearance of zymolyte.It is, for example, possible to use enzymatic determination comes true The activity of the fixed variant.Lipase active can determine by using the p-nitrophenyl (pNP) as described in " example " part It is determined that.
The variant can use methods known in the art to reclaim.For example, can be by a variety of conventional programs from the nutrition The variant is reclaimed in culture medium, these conventional programs include but is not limited to collect, centrifuge, filter, extracting, being spray-dried, steaming Hair is precipitated.
Substantially pure variant, these programs can be obtained come purified variants by multiple programs as known in the art Including but not limited to chromatography is (for example, ion-exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing and size Exclusion chromatography), electrophoretic procedures (for example, preparative isoelectric focusing), differential solubilities (for example, ammonium sulfate precipitation), SDS- PAGE or extraction (see, e.g., protein purification (Protein Purification), Jansen (Janson) and bad are stepped on (Ryden) edit, VCH publishing houses (VCH Publishers), New York, 1989).
On the one hand, the variant is not reclaimed, but the host cell of the invention for expressing the variant is used as the variant Source.
Composition
Consider the composition of the polypeptide including the present invention.In some aspects, the present invention relates to detergent composition, this is washed Wash agent composition and be included in and correspond to SEQ ID NO:Substitution at the E1C and N233C of 2 mature polypeptide position, with fat Fat enzymatic activity, and have at least 60% with the mature polypeptide of parent lipase but be less than 100% sequence identity.
On the one hand, the parent lipase is a kind of lipase, and the lipase is the polypeptide with following amino acid sequence, The amino acid sequence：(a) with the wild type fat from Humicola lanuginosa (Humicola lanuginosa) strain DSM 4109 Enzyme has at least 90% uniformity；(b) compared with the wild type lipase, it is included in E1's or Q249Interior three-dimensional knot Electroneutral or negatively charged amino acid are by the substitution of positively charged amino acid at the surface of structure；In C-terminal include peptide (c) Addition；And/or (d) meets following limitation：(i) negative electrical charge amino acid is included in the position E210 of the wild type lipase； (ii) negatively charged amino acid is included in the region of the position 90-101 corresponding to the wild type lipase；And (iii) include at the position of the N94 corresponding to the wild type lipase neutral or negative electrical charge amino acid and/or corresponding to There is negative net charge or neutral net charge in the position 90-101 of wild type lipase region.
On the one hand, the parent lipase is the lipase with lipase active, the lipase and SEQ ID NO:2 tools There are at least 60% but the sequence identity less than 100%, and corresponding to SEQ ID NO:2 T231R+N233R and At least one or more (for example, several) position in D96E, D111A, D254S, G163K, P256T, G91T, D27R and G38A Putting place includes substitution.
On the one hand, the parent lipase has SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino Acid sequence.On the one hand, the parent lipase includes SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 maturation is more Peptide is made from it.
On the one hand, the present invention relates to the composition comprising lipase Variant, these lipase Variants further comprise Corresponding to SEQ ID NO:Substitution at the position of 2 following any position：2、4、8、11、15、27、33、38、43、48、51、 54、56、57、58、60、69、71、83、86、91、92、94、96、97、98、99、101、111、123、150、152、163、176、 179、187、188、189、198、199、200、210、216、220、224、225、227、228、229、231、236、238、239、 246、249、254、255、256、257、260、263、264、265、266、267、269.On the one hand, these substitutions are selected from SEQ ID NO:2 the following：V2K、Q4R、Q4V、N8R、N11R、Q15C、D27G、D27R、N33K、N33Q、G38A、E43C、 D48C、F51V、S54T、E56K、D57G、S58A、V60K、V60S、L69R、N71C、S83T、I86V、G91A、G91N、G91Q、 N92D、N94K、N94R、D96E、D96G、D96L、D96W、L97M、K98E、K98I、K98Q、E99K、E99N、N101D、N101S、 D111A、T123V、A150G、A152G、G163K、V176L、R179L、V187Y、V187W、Q188R、T189Y、T189W、 H198S、T199R、N200R、E210K、E210Q、S216P、Y220F、S224R、G225R、L227G、L227R、V228R、 P229R、T231R、V236R、I238C、E239C、G246C、Q249R、D254S、I255G、P256K、P256T、P256V、 A257I、A257V、W260C、G263Q、L264A、I265T、G266D、T267A、L269N、L269V.On the one hand, one group of substitution It is selected from：E1C N233C；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K N233C D254S P256T；E1C V2K D27G N33K G38A F51V D96E D111A G163K N233C D254S P256T； E1C V2K D27R N33K G38A F51V D96E D111A G163K Q188R N233C D254S P256T；E1C D27R G38A G91A N92D D96L K98Q D111A G163K N233C D254S P256T；E1C D27R G38A G91N N94R D96E D111A G163K S216P L227G N233C D254S P256T；E1C T231R N233C；E1C T231R N233C Q249R D254S；E1C G225R T231R N233C；E1C Q15C E43C T231R N233C；E1C L227R T231R N233C；E1C P229R T231R N233C；E1C L227G T231R N233C；E1C E99N N101S T231R N233C；E1C L227G T231R N233C D254S；E1C E210K L227G T231R N233C；E1C D27R N33K G38A F51V D96E K98E N101D D111A G163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V S54T E56K D57G L69R D96E K98I D111A A152G G163K T231R N233C D254S P256T；E1C V187Y T189Y L227G T231R N233C；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C V60K I86V A150G E210K L227G T231R N233C P256K；E1C V187W T189W L227G T231R N233C；E1C N94K D96L L227G T231R N233C；E1C G91A N92D D96L K98Q L227G T231R N233C；E1C N8R L227G T231R N233C；E1C L227G V228R T231R N233C；E1C Q4R L227G T231R N233C；E1C N11R L227G T231R N233C；E1C S224R L227G T231R N233C；E1C L227G T231R N233C V236R；E1C N200R L227G T231R N233C；E1C T199R L227G T231R N233C；E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T； E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98I D111AG163K H198S Y220F T231R N233C D254S P256T；E1C D27R N33K G38AF51V E56K L69R D96E K98E D111A G163K R179L T231R N233C D254S P256T A257I；E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T A257I；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T A257I；E1C D27R N33K G38A F51V S54T E56K D57G D96E K98I D111A G163K T231R N233C D254S I255G P256T A257V L269V；E1C V2K D27R N33K G38A F51V L69R D96E K98E D111A G163K V176L E210K L227G T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R G38A F51V L69R D96E K98E D111A G163K E210K T231R N233C D254S P256T；And E1C N11R D27R N33K D48C F51V L69R N71C E87Q K98E N101R T143A E210K G225R L227G P229R T231R N233C Q249R P250R D254S I255G P256K。
On the one hand, compared with parent lipase, the variant has increased stability.
The non-limiting list for the composition component being set forth below is suitable in these compositions, and methods herein In some embodiments that can be eligibly incorporated herein, such as, to aid in or strengthen clean-up performance, need clearly for handling Clean substrate, or to modify the aesthetic feeling of said composition in the case of together with spices, colouring agent, dyestuff or the like.Mix The level for entering any such component in any combinations thing is in addition to the previously cited any material for being used to mix.This The precise nature and its levels of incorporation of a little other components are by depending on the physical form of composition and will be wherein using combination The property of the clean operation of thing.Although being classified according to specific feature to component mentioned below by general heading, Be this and be not construed as limitation because as that will be understood by those of ordinary skill, a kind of component can include other function Property.
Unless otherwise noted, amount in percentage is based on the weight of said composition (wt%).Suitable component material Including but not limited to surfactant, builder, chelating agent, dye transfer inhibitor, dispersant, enzyme and enzyme stabilizers, urge Change material, bleach-activating, hydrogen peroxide, hydrogen peroxide source, pre-formed peracid, polymeric dispersant, clay to remove/resist again Deposition agent, brightener, foam in hibitors, dyestuff, dope dye, spices, perfume delivery system, the agent of structure elastic force, fabric-softening Agent, carrier, hydrotrote, processing aid, solvent and/or pigment.Except disclosed below, the suitable examples of such other components with And use level is found in US 5576282, US6306812 and US 6326348, these documents are combined by quoting This.
Therefore, in certain embodiments, the present invention does not include the one or more in following auxiliary material：Surface-active Agent, soap, builder, chelating agent, dye transfer inhibitor, dispersant, enzyme in addition, enzyme stabilizers, catalysis material, bleach activating Agent, hydrogen peroxide, hydrogen peroxide source, pre-formed peracid, polymeric dispersant, clay removal/anti redeposition agent, brightener, Foam in hibitors, dyestuff, spices, perfume delivery system, the agent of structure elastic force, fabric softener, carrier, hydrotrote, processing are helped Agent, solvent and/or pigment.However, in the presence of one or more components, such one or more components can be following article It is described in detail what ground was present：
Surfactant- surfactant or surfactant system can be included according to the composition of the present invention, wherein The surfactant can be selected from nonionic surface active agent, anion surfactant, cationic surfactant, both sexes Surfactant, zwitterionic surface-active agent, semi-polar nonionic surfactants and its mixture.When it is present, surface Activating agent typically with from 0.1wt% to 60wt%, from 0.2wt% to 40wt%, from 0.5wt% to 30wt%, from 1wt% to 50wt%, from 1wt% to 40wt%, from 1wt% to 30wt%, from 1wt% to 20wt%, from 3wt% to 10wt%, from 3wt% to 5wt%, from 5wt% to 40wt%, from 5wt% to 30wt%, from 5wt% to 15wt%, from 3wt% to 20wt%, From 3wt% to 10wt%, from 8wt% to 12wt%, from 10wt% to 12wt%, from 20wt% to 25wt% or from 25wt%- 60wt% level is present.
Suitable anionic detersive surfactant includes sulfate and sulfonate detersive surfactant.
Suitable sulfonate detersive surfactant includes alkylbenzenesulfonate, is on the one hand C10-13Alkyl benzene sulphonate Salt.Suitable alkylbenzenesulfonate (LAS), suitable LAB bags can be obtained by the commercially available linear alkylbenzene (LAB) of sulfonation (LAB) Include low-carbon 2- phenyl LAB, such as Is °OrOther, which are adapted to LAB, includes high-carbon 2- phenyl LAB, such asSuitable anionic detersive surfactant is the alkylbenzenesulfonate obtained by DETAL Catalytic processes, but Other route of synthesis (such as HF) can also be suitable.On the one hand, using LAS magnesium salts.
Suitable sulphate detersive surfactant includes alkyl sulfate, is C on the one hand8-18Alkyl sulfate, or Predominantly C12Alkyl sulfate.
Other suitable sulphate detersive surfactant is alkyl alkoxylated suifate, is on the one hand alkyl second Epoxide sulfate, is on the one hand C8-18Alkyl alkoxylated suifate, is on the one hand C8-18Alkyl ethoxylated sulfuric acid Salt, typically alkyl alkoxylated suifate have from 0.5 to 20 or from 0.5 to 10 average degree of alkoxylation, typically alkane Alkoxylated sulphate is C8-18Alkyl ethoxylated sulfate, with from 0.5 to 10, from 0.5 to 7, from 0.5 to 5 or from 0.5 to 3 average degree of ethoxylation.
Alkyl sulfate, alkyl alkoxylated suifate and alkylbenzenesulfonate can be straight or branched, substitution or It is unsubstituted.
Detersive surfactant can be the detersive surfactant of middle chain component, on the one hand for middle chain component it is cloudy from The alkylbenzenesulfonate of sub- detersive surfactant, the on the one hand alkyl sulfate for middle chain component and/or middle chain component, example Such as the alkyl sulfate of middle chain component.On the one hand, middle chain component is C1-4Alkyl, typically methyl and/or ethyl.
The non-limiting examples of anion surfactant include sulfate and sulfonate, specifically linear alkylbenzene (LAB) sulphur Hydrochlorate (LAS), LAS isomers, branch-alkylbenzene sulfonate (BABS), phenylalkane sulfonate, alpha-alkene sulfonate (AOS), alkene sulfonate, alkene sulfonates, alkane -2,3- diyls double (sulfate), hydroxy-alkanesulfonates and two sulphurs Hydrochlorate, alkyl sulfate (AS) (such as lauryl sodium sulfate (SDS)), aliphatic alcohol sulfate (FAS), primary alcohol sulfate (PAS), Ether alcohol sulfate (AES or AEOS or FES, also referred to as alcohol ethyoxysulfates or fatty alcohol ether sulphate), secondary alkanesulfonic acid Salt (SAS), paraffin sulfonate (PS), sulfonated ester, the fatty glyceride of sulfonation, α-sulfonic group fatty acid methyl ester (α-SFMe Or SES) (including methyl ester sulfonate (MES)), alkyl succinic acid or alkenyl succinic acid, laurylene base/tetradecene base butanedioic acid (DTSA), the diester and monoesters of the derivative of fatty acid of amino acid, sulfonic group butanedioic acid or soap, and combinations thereof.
Suitable non-ionic detersive surfactant is selected from the group, and the group is made up of the following：C8-C18Alkyl ethoxy Compound, such asC6-C12Alkyl phenol alkoxylates, wherein the alcoxylates unit can be ethyleneoxies Unit, propyleneoxy units or its mixture；C12-C18Alcohol and C6-C12Alkylphenol gathers with ethylene oxide/propylene oxide block The condensation product of compound, such as pluronicC14-C22The alcohol of middle chain component；C14-C22The alkyl of middle chain component Alcoxylates, typically with the average degree of alkoxylation from 1 to 30；Alkyl polysaccharide, is on the one hand APG；It is many Polyhydroxy fatty acid amides；Ether capped poly- (alkoxylate) alcohol surfactant；And its mixture.
Suitable non-ionic detersive surfactant includes APG and/or alkyl alkoxylated alcohol.
On the one hand, non-ionic detersive surfactant includes alkyl alkoxylated alcohol, is on the one hand C8-18Alkyl alkane Epoxide alcohol, such as C8-18Alkyl ethoxylated alcohol, the alkyl alkoxylated alcohol can have from 1 to 50, from 1 to 30, from 1 to 20 or from 1 to 10 average degree of alkoxylation.On the one hand, alkyl alkoxylated alcohol can be C8-18Alkyl ethoxylated alcohol, With from 1 to 10, from 1 to 7, it is more be from 1 to 5 or from 3 to 7 average degree of ethoxylation.Alkyl alkoxylated alcohol can be It is straight or branched and substituted or unsubstituted.Suitable nonionic surfactant includes
The non-limiting examples of nonionic surface active agent include alcohol ethoxylate (AE or AEO), alcohol propoxylation Thing, propenoxylated fatty alcohol (PFA), alkoxylate fatty acid alkyl esters it is (such as ethoxylation and/or propenoxylated Fatty acid alkyl esters), alkylphenol ethoxylate (APE), nonyl phenol ethoxylate (NPE), APG (APG), alkane Epoxide amine, fatty monoethanol amide (FAM), fatty diglycollic amide (FADA), the aliphatic acid monoethanol of ethoxylation The sour acid amides of acid amides (EFAM), propenoxylated fatty monoethanol amide (PFAM), polyhydroxy alkyl fatty or gucosamine N- acyl N-alkyl derivatives (glucamide (GA) or fatty acid glucamides (FAGA)), together with SPAN and TWEEN commodity Under one's name obtainable product, and combinations thereof.
Suitable cationic detersive surfactants include alkyl pyridinium compounds, alkyl quaternary ammonium compound, alkyl quaternary Phosphonium compounds, the sulfonium compound of alkyl three and its mixture.
Suitable cationic detersive surfactants are the quaternary ammonium compounds with below general formula：(R)(R1)(R2)(R3)N+ X-, wherein R is straight or branched, substituted or unsubstituted C6-18Alkyl or alkenyl part, R1And R2Independently selected from methyl or Aminoethyl moiety, R3It is hydroxyl, methylol or hydroxyethyl moieties, X is to provide the anion of neutral charge, and suitable anion includes Halide, such as chloride；Sulfate；And sulfonate.Suitable cationic detersive surfactants are single-C6-18Alkyl List-hydroxyethyl dimethyl aliquat.Highly suitable cationic detersive surfactants are single-C8-10Alkyl list-hydroxyl second Base dimethyl quaternary ammonium chloride, single-C10-12Alkyl list-hydroxyethyl dimethyl aliquat and single-C10Alkyl list-hydroxyl second Base dimethyl quaternary ammonium chloride.
The non-limiting examples of cationic surfactant include alkyl dimethyl ethanol quaternary amine (ADMEAQ), cetyl Trimethylammonium bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC) and alkyl benzyl dimethyl ammonium, quaternary ammonium alkyl Compound, alkoxy quaternary ammonium (AQA) compound, ester quat and combinations thereof.
Suitable amphoteric surfactant/zwitterionic surface-active agent includes amine oxide and glycine betaine (such as alkyl-dimethyl Base glycine betaine, sulfobetaines) or its combination.Anion surfactant-anion surface active that the amine of the present invention is neutralized The anionic cosurfactants of agent and auxiliary can be existed by sour form, and the sour form can be neutralized to be formed It is desirably used for the surfactant salt of detergent composition of the present invention.The reagent for being typically used for neutralizing includes metal counter ion Alkali, such as hydroxide, such as NaOH or KOH.For neutralizing the anion surfactant of the present invention and in its sour form Aiding in the other preferred reagent of anion surfactant or cosurfactant includes ammonia, amine or alkanolamine.It is preferred that alkanol Amine.Suitable non-limiting examples include MEA, diethanol amine, triethanolamine and other are as known in the art straight The alkanolamine of chain or side chain；For example, highly preferred alkanolamine includes 2- amino -1- propyl alcohol, 1- aminopropanols, single isopropanol Amine or 1- amino -3- propyl alcohol.Amine neutralizes and may proceed to complete or partial degree, for example, anion surfactant is mixed The part of thing can be neutralized by sodium or potassium, and the part of anionic surfactant mixture can be by amine or alkanolamine With.
The non-limiting examples of Semi-polar surfactants include amine oxide (AO), such as alkyldimethylamine oxide
Comprising one or more anion surfactants and another or a variety of nonionic surfactants and can Can be excellent optionally with the surfactant system of the mixture of other surfactant such as cationic surfactant Choosing.It is preferred that anion and the weight ratio of nonionic surfactant be at least 2:1 or at least 1:1 to 1:10.
In one aspect, surfactant system may include by the isoprenoid table of chemical formula A and chemical formula B representatives A kind of mixture of face activating agent：
Wherein Y is CH2Or it is empty, and Z can be selected such that obtained by surfactant be to be selected from following table Face activating agent：The many alcoxyl based surfactants of alkyl carboxylic acid ester surfactant, alkyl, many alkoxy sulfuric acid of alkyl anion type It is ester surfactant, alkyl glycerol ester sulfonate ester surfactant, alkyl dimethyl amine oxide surfactant, many based on alkyl Surfactant, alkyl phosphoric acid ester surfactant, alkyl glycerol sulfonates surfactant, many gluconates of alkyl of hydroxyl Surfactant, alkyl polyphosphate surfactant, alkyl phosphonate surfactant, alkyl polyglucoside surfactant, Alkyl monoglycosides surfactant, alkyl bioside surfactant, alkyl sulfosuccinates surfactant, the sulphur of alkyl two Acid ester surface active agent, alkyl disulfonate surfactant, alkyl sulfosuccinate amic acid esters surfactant, alkyl grape Sugared acidamide surfactant, alkyl taurine ester surfactant, alkylsarcosines ester surfactant, alkyl glycine ester table Face activating agent, alkyl hydroxyethyl sulfonate ester surfactant, the alkanol amide surfactants of alkyl two, alkyl mono-alkanol acyl Amine surfactant, alkyl monoalkanolamide sulfuric acid ester surfactant, alkyl dihydroxy yl acetamide surfactant, alkyl Dihydroxy yl acetamide sulfuric acid ester surfactant, alkyl glycerol ester surfactant, alkyl glycerol ester sulfuric acid ester surfactant, Alkyl glycerol ether surface active agent, alkyl glycerylether sulfuric acid ester surfactant, alkyl methacrylate ester sulfonate ester surfactant, alkane Quito glycerine ether surface active agent, alkyl poly-glycerine ether sulfuric ester surfactant, alkyl sorbitan ester surfactant, alkane Base amino alkane sulfonate ester surfactant, alkylamide propyl-betaine surfactant, based on polyoxyethylene base quaternary ammonium The surfactant of salt, the surfactant based on alkyl monohydroxy alkyl-di-alkyl quaternary ammonium salt, based on alkyl bis-hydroxy The surfactant of alkyl monoalkyl quaternary ammonium salts, alkylated quaternary ammonium salt surfactant, alkyl trimethyl quaternized ammonium surface are lived Property agent, the surfactant based on alkyl polyhydroxy alkyl oxy quaternary ammonium salts, alkyl glycerol Esterquat Surfactant, Alkyl glycol amine quaternary surfactant, alkyl monomethyl dihydroxy ethyl quaternary surfactant, alkyl dimethyl list Hydroxyethyl quaternary surfactant, alkyl trimethyl ammonium surfactant, the surfactant based on alkyl imidazoline, alkene Hydrocarbon -2- bases-succinate surfactant, alkyl a- sulfonate carboxylic acids surfactant, alkyl a- sulfonate carboxylic acid's Arrcostabs surface Activating agent, alhpa olefin sulfonate ester surfactant, alkyl phenol ethoxylate surfactant, alkyl benzene sulfonic acid ester surface are lived Property agent, alkyl sulfo betaines surfactant, alkyl hydroxy sulfobetaine surfactant, alkyl ammonium group carboxylic acid glycine betaine The single alkane of surfactant, alkyl sucrose ester surfactant, alkylalkanol acidamide surfactant, alkyl two (polyethylene glycol oxide) Base ammonium surfactant, alkyl list (polyethylene glycol oxide) dialkyl ammonium surfactant, alkyl benzyl dimethyl ammonium surface-active Agent, alkyl aminopropionic acid ester surfactant, alkylamide propyl dimethylamine surfactant or a kind of its mixture；And If Z is live part, Z is by a kind of suitable metal or Organic counter-ion by charge balance.Suitable counter ion includes Metal counter ion, amine or alkanolamine, such as C1-C6 alkanol ammoniums.More precisely, suitable counter ion includes Na+, Ca+, Li +, K+, Mg+, such as MEA (MEA), diethanol amine (DEA), triethanolamine (TEA), 2- amino-l- propyl alcohol, 1- aminopropans Alcohol, methyl diethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, l- amino -3- propyl alcohol or its mixture. On the one hand, these compositions include the one or more non-isoprenoid surfactants and one kind from 5% to 97% Or a variety of secondary additives；The weight of surfactant and the surfactant with chemical formula B wherein with chemical formula A It is from 50 to measure ratio:50 to 95:5.
Soap-composition in this can include soap.Without being limited by theory, it can it is desirable to include soap, because it Part serves as surfactant and builder is served as in part, and available for suppressing foam, and furthermore, it is possible to advantageously with A variety of cationic compounds of composition interact to strengthen the flexibility of the textile fabric handled with the present composition. Any soap as known in the art for being used in laundry detergent compositions can be utilized.In one aspect, these composition bags Containing the soap from 0wt% to 20wt%, from 0.5wt% to 20wt%, from 4wt% to 10wt% or from 4wt% to 7wt%.
The example of useful soap includes oleate soap, palmitic acid soap, palm kernel fatty acid soap and its mixture herein.Typical case Soap be in the form of the fatty acid soaps mixture with different chain length and substitution value.A kind of such mixture is topping palm Benevolence aliphatic acid.
On the one hand, the soap is selected from free fatty acids.Suitable aliphatic acid is saturation and/or undersaturated and can be from Natural origin such as vegetable esters or animal ester (for example, palm-kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tall oil, Castor oil, butter and fish oil, grease and its mixture) middle acquisition, or it is synthetically prepared (for example, oxidation or warp via oil By fischer tropsch process (Fisher Tropsch pr °C ess) by the hydrogenation of carbon monoxide).
The example of suitable saturated fatty acid for using in the compositions of the present invention includes capric acid, laurate, Pork and beans Cool acid, palmitic acid, stearic acid, arachidic acid are He behenic acid.Suitable unrighted acid species includes：Palmitoleic acid, oleic acid, Asia Oleic acid, leukotrienes and castor oil acid.It is preferred that the example of aliphatic acid be saturation Cn aliphatic acid, saturation Ci2-Ci4Aliphatic acid and full And/or undersaturated Cn to Ci8Aliphatic acid and its mixture.
When it is present, the weight of fabric-softening cation cosurfactant and aliphatic acid is than preferably from about 1:3 To about 3:1, more preferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.
The level of soap and non-soap anionic surfactant in this be with acid basis specify by detergent composition Weight meter percentage.However, as being generally understood that in this area, in practice using sodium, potassium or alkanol ammonium alkali such as With anion surfactant and soap in sodium hydroxide or MEA.
Water-assisted solventThe composition of-the present invention can include one or more water-assisted solvents.Water-assisted solvent is following chemical combination Thing, the compound dissolves hydrophobic compound (or on the contrary, polar substances) in nonpolar environment in aqueous solution.Typical case Ground, water-assisted solvent has hydrophilic and hydrophobic two kinds of features (such as from surfactant known so-called amphiphilic nature)；But help water The molecular structure of solvent is typically unfavorable for spontaneous self aggregation, and (Kaler) is strangled see, for example, Huo Qideng (Hodgdon) and card (2007), colloid is newly shown in (Current Opinion in Colloid＆Interface Science), 12 with interface science: 121-128 summary.Water-assisted solvent does not show critical concentration, will occur such as institute for Surfactant higher than the concentration It was found that self aggregation and lipid formation micella, thin layer or other well-defined interphases.On the contrary, many water-assisted solvents are shown The accumulation process of continuous type, the wherein size of aggregation increase as concentration increases.However, many water-assisted solvents are changed System (mixture for including water, oil, surfactant and polymer) comprising polarity and the material of apolar character mutually goes For, stability and colloid property.Classical ground uses water-assisted solvent from pharmacy, personal nursing, inter-trade applied to technology of food. Water-assisted solvent use in detergent compositions allows for example denseer surfactant formulatory product (such as by going to remove water During compressed liquid detergent) without causing undesirable phenomenon, for example it is separated or high viscosity.
Detergent can include from 0 to 10wt%, such as from 0 to 5wt%, 0.5wt% to 5wt% or from 3wt% to 5wt% water-assisted solvent.Any water-assisted solvent as known in the art for being used in detergent can be utilized.Help water-soluble The non-limiting examples of agent include benzene sulfonic acid sodium salt, paratoluenesulfonic acid sodium salt (STS), sodium xylene sulfonate (SXS), cumene sodium sulfonate (SCS), cymene sodium sulfonate, amine oxide, alcohol and polyglycol ether, hydroxynaphthoic acid sodium, croceine acid sodium, ethylhexyl sulphur Sour sodium and combinations thereof.
BuilderThe composition of-the present invention can include one or more builders, co-builder, builder system or its Mixture.When a builder is used, Cleasing compositions will typically comprise from 0 to 65wt%, at least 1wt%, from 2wt% to 60wt% or the builder from 5wt% to 10wt%.In washing tableware Cleasing compositions, the level of builder is typically 40wt% to 65wt% or 50wt% to 65wt%.Said composition can be substantially free of builder；Substantially free of meaning " not intentionally adding " zeolite and/or phosphate.Typical zeolite builders include Wessalith CS, zeolite P and zeolite MAP.Typical case Phosphate builder be sodium tripolyphosphate.
Builder and/or co-builder can be specifically the chelating for forming the water-soluble compound with Ca and Mg.Can To use any builder and/or co-builder for being used to use in detergent as is generally known in the art.Builder it is unrestricted Property example include zeolite, diphosphate (pyrophosphate), triphosphate such as sodium tripolyphosphate (STP or STPP), carbonate for example Sodium carbonate, soluble silicate such as sodium metasilicate, phyllosilicate (such as SKS- from Hirst company (Hoechst) 6), monoethanolamine such as 2- amino second -1- alcohol (MEA), diethanolimine (DEA) and 2,2 ', 2 "-nitrilotriethanol (TEA), And Carboxymethylinulin (CMI), and combinations thereof.
Cleasing compositions can individually comprise co-builder, or be combined with builder (such as zeolite builders).Help altogether The non-limiting examples of lotion include the homopolymer or its copolymer of polyacrylate, such as poly- (acrylic acid) (PAA) or copolymerization (acrylic acid/maleic acid) (PAA/PMA).Other non-limiting examples include citrate, chelating agent (such as amino carboxylic acid Salt, aminopolycanboxylic acid's salt and phosphate) and alkyl-or alkenyl succinic acid.Other instantiation includes 2,2 ', 2 "-secondary nitrogen Base triacetic acid (NTA), ethylenediamine tetra-acetic acid (EDTA), diethylene-triamine pentaacetic acid (DTPA), imido disuccinic acid (IDS), Ethylenediamine-N, N '-disuccinic acid (EDDS), MDGA (MGDA), glutamic acid-N, N- oxalic acid (GLDA), 1- Hydroxyl ethane -1,1- diyls double (phosphonic acids) (HEDP), ethylenediamine tetraacetic (methylene) four (phosphonic acids) (EDTMPA), diethylenetriamines The single acetic acid of five (methylene) five (phosphonic acids) (DTPMPA), N- (2- ethoxys) iminodiacetic acid (EDG), aspartic acid-N- (ASMA) the single propionic acid (ASMP) of, aspartic acid-N, N- oxalic acid (ASDA), aspartic acid-N-, imido disuccinic acid (IDA), N- (2- sulphurs methyl) aspartic acid (SMAS), N- (2- sulfoethyls) aspartic acid (SEAS), N- (2- sulphurs methyl) glutamic acid (SMGL), N- (2- sulfoethyls) glutamic acid (SEGL), N- methyliminodiacetic acids (MIDA), α-alanine-N, N- oxalic acid (α- ALDA), serine-N, N- oxalic acid (SEDA), isoerine-N, N- oxalic acid (ISDA), phenylalanine-N, N- oxalic acid (PHDA), ortho-aminobenzoic acid-N, N- oxalic acid (ANDA), p-aminobenzene sulfonic acid-N, N- oxalic acid (SLDA), taurine-N, N- oxalic acid (TUDA) and sulphur methyl-N, N- oxalic acid (SMDA), N- (ethoxy)-ethylene amine triacetic acid (HEDTA), two Ethanol glycine (DEG), diethylene triamine penta(methylene phosphonic acid) (DTPMP), amino three (methylene phosphonic acid) (ATMP) and It is combined and salt.Other exemplary builder and/or co-builder is described in such as WO 09/102854, US 5977053 In.
On the one hand, the present invention relates to composition, these compositions include the variant of parent lipase, and the variant is included in Corresponding to SEQ ID NO:Substitution at the E1C and N233C of 2 mature polypeptide position, with lipase active, and with SEQ ID NO:2 mature polypeptide has at least 60% but the sequence identity less than 100%, and said composition is comprising at most 10wt% or 15wt% alumino-silicate (anhydrous) and/or phosphate builder, said composition has the deposit more than 4 or 7.5 Basicity.As used herein, term " reserve alkalinity " is i.e. in order to calculate reserve alkalinity by using the 1% of HCI composition (w/v) measurement (g/NaOH/100g compositions) of the buffer capacity of said composition determined by solution to pH 7.5.Can such as it exist The upper disclosed calculating reserve alkalinity of page 9 in WO 2006/090335.On the one hand, the parent lipase is a kind of fat Enzyme, the lipase is the polypeptide with following amino acid sequence, the amino acid sequence：(a) with being derived from Humicola lanuginosa The wild type lipase of (Humicola lanuginosa) strain DSM 4109 has at least 90% uniformity；(b) with it is described Wild type lipase is compared, and is included in E1's or Q249Electroneutral or negatively charged amino at the surface of interior three-dimensional structure Acid is by the substitution of positively charged amino acid；In C-terminal including peptide add (c)；And/or (d) meets following limitation：(i) exist The position E210 of the wild type lipase includes negative electrical charge amino acid；(ii) in the position corresponding to the wild type lipase Putting 90-101 region includes negatively charged amino acid；And (iii) is the N94's corresponding to the wild type lipase Include neutral or negative electrical charge amino acid at position and/or in the region corresponding to the position 90-101 of the wild type lipase With negative net charge or neutral net charge.On the one hand, the parent lipase is the lipase with lipase active, with SEQ ID NO:2 have at least 60% but the sequence identity less than 100%, and corresponding to SEQ ID NO:2 T231R+ In N233R and D96E, D111A, D254S, G163K, P256T, G91T, D27R and G38A at least one or more (for example, Several) include substitution at position.On the one hand, the parent lipase has SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino acid sequence.On the one hand, the parent lipase includes SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide is made from it.On the one hand, the present invention relates to the composition comprising lipase Variant, these lipase Variant further comprises corresponding to SEQ ID NO:Substitution at the position of 2 following any position：2、4、8、11、15、27、 33、38、43、48、51、54、56、57、58、60、69、71、83、86、91、92、94、96、97、98、99、101、111、123、 150、152、163、176、179、187、188、189、198、199、200、210、216、220、224、225、227、228、229、 231、236、238、239、246、249、254、255、256、257、260、263、264、265、266、267、269.On the one hand, These substitutions are selected from SEQ ID NO:2 the following：V2K、Q4R、Q4V、N8R、N11R、Q15C、D27G、D27R、N33K、 N33Q、G38A、E43C、D48C、F51V、S54T、E56K、D57G、S58A、V60K、V60S、L69R、N71C、S83T、I86V、 G91A、G91N、G91Q、N92D、N94K、N94R、D96E、D96G、D96L、D96W、L97M、K98E、K98I、K98Q、E99K、 E99N、N101D、N101S、D111A、T123V、A150G、A152G、G163K、V176L、R179L、V187Y、V187W、Q188R、 T189Y、T189W、H198S、T199R、N200R、E210K、E210Q、S216P、Y220F、S224R、G225R、L227G、 L227R、V228R、P229R、T231R、V236R、I238C、E239C、G246C、Q249R、D254S、I255G、P256K、 P256T、P256V、A257I、A257V、W260C、G263Q、L264A、I265T、G266D、T267A、L269N、L269V.One Aspect, one group of substitution is selected from：E1C N233C；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K N233C D254S P256T；E1C V2K D27G N33K G38A F51V D96E D111A G163K N233C D254S P256T；E1C V2K D27R N33K G38A F51V D96E D111A G163K Q188R N233C D254S P256T；E1C D27R G38A G91A N92D D96L K98Q D111A G163K N233C D254S P256T；E1C D27R G38A G91N N94R D96E D111A G163K S216P L227G N233C D254S P256T；E1C T231R N233C；E1C T231R N233C Q249R D254S；E1C G225R T231R N233C；E1C Q15C E43C T231R N233C；E1C L227R T231R N233C；E1C P229R T231R N233C；E1C L227G T231R N233C；E1C E99N N101S T231R N233C；E1C L227G T231R N233C D254S；E1C E210K L227G T231R N233C；E1C D27R N33K G38A F51V D96E K98E N101D D111AG163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V S54T E56K D57G L69R D96E K98I D111A A152G G163K T231R N233C D254S P256T；E1C V187Y T189Y L227G T231R N233C； E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C V60K I86V A150G E210K L227G T231R N233C P256K；E1C V187W T189W L227G T231R N233C；E1C N94K D96L L227G T231R N233C；E1C G91A N92D D96L K98Q L227G T231R N233C；E1C N8R L227G T231R N233C；E1C L227G V228R T231R N233C；E1C Q4R L227G T231R N233C；E1C N11R L227G T231R N233C；E1C S224R L227G T231R N233C；E1C L227G T231R N233C V236R；E1C N200R L227G T231R N233C；E1C T199R L227G T231R N233C；E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98I D111A G163K H198S Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V E56K L69R D96E K98E D111A G163K R179L T231R N233C D254S P256T A257I；E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T A257I；E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T A257I；E1C D27R N33K G38A F51V S54T E56K D57G D96E K98I D111A G163K T231R N233C D254S I255G P256T A257V L269V；E1C V2K D27R N33K G38A F51V L69R D96E K98E D111A G163K V176L E210K L227G T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T； E1C D27R G38A F51V L69R D96E K98E D111A G163K E210K T231R N233C D254S P256T； And E1C N11R D27R N33K D48C F51V L69R N71C E87Q K98E N101R T143A E210K G225R L227G P229R T231R N233C Q249R P250R D254S I255G P256K。
Chelating agent and crystal growth inhibitor- composition in this can include chelating agent and/or crystal growth inhibitor. Suitable molecule includes copper, ion and/or manganese chelating agent and its mixture.Suitable molecule includes DTPA (diethylenetriamines Pentaacetic acid), HEDP (hydroxyethane diphosphonic acid), DTPMP (diethylene triamine penta(methylene phosphonic acid)), 1,2- dihydroxy benzenes- 3,5- disulfonates salt hydrate, ethylenediamine, diethylenetriamines, ethylenediamine disuccinic acid (EDDS), N- hydroxyethyl second Ethylenediamine triacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N- hydroxyethyliminodiacetic acids (HEIDA), dihydroxy Base ethyl glycine (DHEG), the propionic acid of ethylene diamine four (EDTP), Carboxymethylinulin and 2- phosphinylidyne butanes 1,2,4- tri- Carboxylic acid () and its derivative AM.Typically, said composition can include from 0.005wt% to 15wt% or from 3.0wt% to 10wt% chelating agent or crystal growth inhibitor.
Bleaching component- the bleaching component being suitable in the method and composition of the incorporation present invention includes more than one bleaching One kind or mixture in component.Suitable bleaching component includes bleaching catalyst, optical white, bleach-activating, peroxidating Hydrogen, hydrogen peroxide source, pre-formed peracid and its mixture.Generally, when using bleaching component, composition of the invention can be with Including from 0 to 30wt%, from 0.00001wt% to 90wt%, 0.0001wt% to 50wt%, from 0.001wt% to 25wt% Or from 1wt% to 20wt%.The example of suitable bleaching component includes：
(1) pre-formed peracid：Suitable pre-formed peracid includes but is not limited to the compound being selected from the group, the group by The following is constituted：Pre-formed peroxy acid or its salt, typically peroxycarboxylic acid or its salt or peroxosulphuric or its salt.
Pre-formed peroxy acid or its salt are preferably peroxycarboxylic acid or its salt, typically with corresponding to below formula Chemical constitution：
Wherein：R14Selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic group；R14Group can be straight or branched , it is substituted or unsubstituted；And Y is any suitable counter ion for realizing neutral charge, it is preferable that Y is selected from hydrogen, sodium or potassium. Preferably, R14It is straight or branched, substituted or unsubstituted C6-9Alkyl.Preferably, peroxy acid or its salt be selected from peroxide oneself Acid, peroxide enanthic acid, Peroxycaprylic acid, pernoanoic acid, peroxydecanoic and its salt, or its any combinations.Particularly preferred peroxy acid is Phthalimide-based-peroxy-alkanoic acid, particularly ε-phthalimide-based peroxy caproic acid (PAP).It is preferred that Ground, peroxy acid or its salt have the fusing point in the range of from 30 DEG C to 60 DEG C.
Pre-formed peroxy acid or its salt can also be peroxosulphuric or its salt, typically with corresponding to below formula Chemical constitution：
Wherein：R15Selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic group；R15Group can be straight or branched , it is substituted or unsubstituted；And Z is any suitable counter ion for reaching neutral charge, it is preferable that Z be selected from hydrogen, sodium or Potassium.Preferably, R15It is straight or branched, substituted or unsubstituted C6-9Alkyl.Preferably, such bleaching component can by from Amounts of the 0.01wt% to 50wt% or from 0.1wt% to 20wt% is present in the composition of the present invention.
(2) hydrogen peroxide source includes such as inorganic perhydrate salts, including alkali metal salt, such as perborate (generally Monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, the sodium salt and its mixture of persilicate. An aspect of of the present present invention, inorganic perhydrate salts are those for being selected from the following group, and the group is made up of the following：Cross boron The sodium salt and its mixture of hydrochlorate, percarbonate.When deployed, inorganic perhydrate salts are typically with entire combination thing 0.05wt% to 40wt% or 1wt% to 30wt% amount exists and is typically impregnated in such composition as can be by The crystalline solid of coating.Suitable coating includes：Inorganic salts, such as alkali silicate, carbonate or borate or its mixing Thing, or organic material, such as water-soluble or aqueous dispersion polymers, wax, oil or fat soap.Preferably, such bleaching component can In the composition for being present in the present invention with the amount by 0.01wt% to 50wt% or 0.1wt% to 20wt%.
(3) term bleach-activating means via hydrolysis is crossed with hydroperoxidation to form the chemical combination of peracid herein Thing.The peracid formed in this way constitutes the bleaching agent of activation.Suitable bleach-activating to be used includes belonging to ester, acyl herein Amine, acid imide or anhydrides it is other those.Suitable bleach-activating is those with R- (C=O)-L, and wherein R is alkyl Group (being optionally side chain), when the bleach-activating is hydrophobic, with the carbon atom from 6 to 14 or from 8 To 12 carbon atoms, and when the bleach-activating is hydrophilic, having less than 6 carbon atoms or less than 4 carbon atoms； And it is L leaving groups.The example of suitable leaving group is the benzene sulfonate of benzoic acid and its derivative-especially.Suitable Bleach-activating includes lauroyl epoxide benzene sulfonate, decanoyloxybenzenesulphonate, decanoyloxybenzoic acid or its salt, 3,5,5- Trimethyl acetyl epoxide benzene sulfonate, tetra acetyl ethylene diamine (TAED), 4- [(3,5,5- trimethyl acetyls base) epoxide] benzene -1- Sodium sulfonate (ISONOBS), 4- (dodecanoyl epoxide) benzene -1- sulfonate (LOBS), 4- (capryl epoxide) benzene -1- sulfonate, 4- (capryl epoxide) benzoate (DOBS or DOBA), 4- (pelargonyl group epoxide) benzene -1- sulfonate (NOBS)), and/or disclose Those in WO 98/17767.The family of bleach-activating is disclosed in EP 624154 and especially excellent in that family Choosing is ATEC (ATC).There is it to be environment-friendly excellent for ATC or short chain triglyceride (as glyceryl triacetate) Point.In addition, ATEC and glyceryl triacetate have good hydrolytic stability in the product in storage, and it is to have The bleach-activating of effect.Finally, ATC is multi-functional, because the citrate that discharges can be as helping in hydrolysis is crossed Lotion works.Alternately, bleaching system can include the peroxy acid of such as acid amides, acid imide or sulfone type.The bleaching system Peracid, such as 6- (phthalimido) peracetic acid (PAP) can also be included.Suitable bleach-activating is also disclosed in WO 98/ In 17767.Although any suitable bleach-activating, in one aspect of the invention, theme Cleasing compositions can be used NOBS, TAED or its mixture can be included.When it is present, based on fabric and household care composition, peracid and/or bleaching are lived Agent is generally present in composition with 0.1wt% to 60wt%, 0.5wt% to 40wt% or 0.6wt% to 10wt% amount. One or more hydrophobicity peracid or its precursor can be used with one or more hydrophilic peracids or its combination of precursors.It is preferred that Ground, such bleaching component can be present in the combination of the present invention by 0.01wt% to 50wt% or 0.1wt% to 20wt% amount In thing.
The amount of hydrogen peroxide source and peracid or bleach-activating can be selected, to cause available oxygen (to come from peroxide Compound source) with the mol ratio of peracid it is from 1:1 to 35:1, or even 2:1 to 10:1.
(4) diacyl peroxide-preferred diacyl peroxide bleaching species includes being selected from below general formula Those of diacyl peroxide：R1-C(O)-OO-(O)C-R2, wherein R1Represent C6-C18Alkyl, preferably comprising with extremely Lack the straight chain of 5 carbon atoms and optionally include one or more substituents (such as-N+(CH3)3,-COOH or-CN) and/or The C of the one or more interrupt units (such as-CONH- or-CH=CH-) inserted between the adjacent carbon atom of alkyl6-C12Alkane Base group, and R2Represent and the compatible aliphatic group of peroxide portion, to cause R1And R2Together comprising 8 altogether To 30 carbon atoms.In a preferred aspect, R1And R2It is the unsubstituted C of straight chain6-C12Alkyl chain.Most preferably, R1And R2 It is identical.Diacyl peroxide (wherein R1And R2It is C6-C12Alkyl group) it is particularly preferred.Preferably, R group (R1Or R2) at least one, most preferably only have one in α not comprising branch or sagging rings, or preferably at α or β all not comprising branch or sagging rings, or most preferably in α or β or γ all not comprising branch or sagging rings. In a further preferred embodiment, DAP can be asymmetric, to cause the hydrolysis of preferably R1 carboxyl groups to be fast Speed to produce peracid, but the hydrolysis of R2 carboxyl groups is slow.
Four acyl peroxides bleaching species is preferably selected from four acyl peroxides with below general formula：R3-C(O)- OO-C(O)-(CH2)n-C(O)-OO-C(O)-R3, wherein R3Represent C1-C9Alkyl, or C3-C7Group, and n represented from 2 to 12 Or the integer of 4 to 10 (including end value).
Preferably, diacyl and/or four acyl peroxides bleaching species with enough provide by weight at least 0.5ppm, The amount of at least 10ppm or at least 50ppm cleaning solution is present.In a preferred embodiment, these bleach species to carry enough Amount for the cleaning solution by weight from 0.5ppm to 300ppm, from 30ppm to 150ppm is present.
Preferably, bleaching component includes bleaching catalyst (5 and 6).
(5) preferably organic (nonmetallic) bleaching catalyst, including the oxygen from peroxy acid and/or its salt can be received Atom and by the bleaching catalyst of the Oxygen atom transfer to oxidable substrate.Suitable bleaching catalyst includes but not limited In：Iminium cations and polyion；Imines hybrid ion；Modified amine；Modified oxidized amine；N- sulphonyl imines；N- phosphonos Base imines；N- acyl imines；Thiadiazoles dioxide；Perfluor imines；Cyclic sugar and its mixture.
Suitable iminium cations and polyion include but is not limited to, N- methyl -3,4- dihydro-isoquinoline tetrafluoro boron Hydrochlorate, such as tetrahedron (Tetrahedron) (1992), 49 (2), the progress described in 423-38 prepare (such as compound 4, the 433rd Page)；The p- toluene fulfonate of N- methyl -3,4- dihydro-isoquinolines, the progress as described in US 5360569 prepares the (the such as the 11st Column, example 1)；And the p- toluene fulfonate of n-octyl -3,4- dihydro-isoquinoline, it is prepared by the progress as described in US5360568 (such as the 10th column, example 3).
Suitable imines hybrid ion includes but is not limited to, N- (3- sulfopropyls) -3,4- dihydro-isoquinolines, inner salt, Progress as described in US 5576282 is prepared on (such as the 31st column, example II)；N- [2- (sulphur epoxide) dodecyl] -3,4- dihydros Isoquinolin, inner salt, the progress as described in US 5817614 is prepared on (for example, the 32nd column, example V)；2- [3- [(2- ethyl hexyls Base) epoxide] -2- (sulphur epoxide) propyl group] -3,4- dihydro-isoquinolines, it is prepared by inner salt, the progress as described in WO 05/047264 (for example, page 18, example 8), and 2- [3- [(2- butyl octyls) epoxide] -2- (sulphur epoxide) propyl group] -3,4- dihydro isoquinolines Quinoline, inner salt.
Suitable modified amine oxygen transfer catalyst includes but is not limited to 1,2,3,4- tetrahydrochysene -2- methyl isophthalic acids-isoquinolin alcohol, its Can be according to the method system being described in Tet Lett (Tetrahedron Letters) (1987), 28 (48), 6061-6064 It is standby.Suitable modified oxidized amine oxygen transferring catalyst include but is not limited to 1- hydroxy-ns-epoxide-N- [2- (sulphur epoxide) decyl]- 1,2,3,4- tetrahydroisoquinoline sodium.
Suitable N- sulphonyl imine oxygen transferring catalyst includes but is not limited to according to Journal of Organic Chemistry (Journal of Organic Chemistry) (1990), 55 (4), 3- methyl isophthalic acids prepared by the program described in 1254-61,2- benzisothiazoles 1,1- dioxide.
Suitable N- phosphonyl imine oxygen transfer catalysts include but is not limited to, [R- (E)]-N- [(chloro- 5- nitrobenzene of 2- Base) methylene]-to phenyl-p- (2,4,6- trimethylphenyl) phosphinic acid amide, it can be according to being described in Chemical Society's magazine (Journal of the Chemical S °C iety), chemical communication (Chemical Communications) (1994), (22), prepared by the method in 2569-70.
Suitable N- acyl imine oxygen transferring catalysts include but is not limited to can be according to Polish The Chemicals (Polish Journal of Chemistry) (2003), 77 (5), [N (E)]-N- (phenyl methylidenes of the program manufacture described in 577-590 Base) acetamide.
Suitable thiadiazoles dioxide oxygen transferring catalyst include but is not limited to can according to US 5753599 (the 9th column, Example 2) described in program manufacture 3- methyl 4-phenyl -1,2,5- thiadiazoles 1,1- dioxide.
Suitable perfluor imines oxygen transfer catalyst includes but is not limited to fluoro- N- (the nonyl fluorine of (Z) -2,2,3,3,4,4,4- seven Butyl) butyryl imines fluoride, its can according to Tet Lett (Tetrahedron Letters) (1994), 35 (34), It is prepared by the method described in 6329-30.
Suitable cyclic sugar oxygen transferring catalyst includes but is not limited to such as in US 6649085 (the 12nd column, example 1) 1,2 prepared:Red -2,3- acetyl butyryls (the hexodiuro) -2,6- pyranoses of-O- isopropylidenes-D- of 4,5- bis-.
Preferably, bleaching catalyst includes imines and/or carbonyl functional group and typically when receiving oxygen atom, especially It is can to form phenoxy imine cation (oxaziridinium) when receiving the oxygen atom from peroxy acid and/or its salt And/or dioxirane functional group.Preferably, bleaching catalyst includes phenoxy imine cation functional group and/or is receiving oxygen Atomic time, especially it can form phenoxy imine cation functional group when receiving the oxygen atom from peroxy acid and/or its salt. Preferably, bleaching catalyst includes cyclic imide functional group, and the annulus has from five to eight atom preferably wherein The ring size of (including nitrogen-atoms), preferably six atoms.Preferably, bleaching catalyst includes arylimine functional groups, preferably Aryl bicyclic imine, preferably 3,4- dihydro-isoquinolines functional group.Typically, imine is season imines function Roll into a ball and typically when receiving oxygen atom, can especially be formed when receiving the oxygen atom from peroxy acid and/or its salt Season phenoxy imine cation functional group.On the one hand, the detergent composition includes with no more than 0, is not more than -0.5, less In -1.0, no more than -1.5, no more than -2.0, no more than -2.5, no more than -3.0 or no more than -3.5 logPo/wBleaching Component.Describe in more detail below for determining logPo/wMethod.
Typically, bleach can be produced with from 0.01 to 0.30, from 0.05 to 0.25 or from 0.10 to 0.20 XSOBleaching species.Describe in more detail below for determining XSOMethod.For example, the bleaching with isoquinoline structure Composition can produce the bleaching species with phenoxy imine cation structure.In this example, XSOIt is phenoxy imine cation Bleach the X of speciesSO。
Preferably, bleaching catalyst has the chemical constitution corresponding to below formula：
Wherein：N and m are independently that preferably n and m are 0 from 0 to 4；Each R1Independently selected from substituted or unsubstituted The group being selected from the group, the group is made up of the following：Hydrogen, alkyl, cycloalkyl, aryl, fused-aryl, heterocycle, fusion are miscellaneous Ring, nitro, halogen, cyano group, sulfonate radical, alkoxy, ketone group, carboxyl and alkoxy carbonyl group；And the R of any two vicinal1Take It can merge to form fused-aryl, fused iso or annelated heterocycles for base；Each R2Independently selected from substituted or unsubstituted The group independently selected from the following group, the group is made up of the following：Hydrogen, hydroxyl, alkyl, cycloalkyl, alkaryl, aryl, virtue Alkyl, alkylidene, heterocycle, alkoxy, aryl carbonyl, carboxyalkyl and amide group；Any R2Can be with any other R2 It is combined together to form a part for common ring；It is any together with R2It can merge to form carbonyl；And any two R2It can close And to form the unsaturated part of substituted or unsubstituted fusion；R3It is C1To C20Substituted or unsubstituted alkyl；R4It is hydrogen Or Qt- part A, wherein：Q is that the alkene of branch or non-branch, t=0 or 1, and A are the anionic groups being selected from the group, should Group is made up of the following：OSO3 -、SO3 -、CO2 -、°CO2 -、OPO3 2-、OPO3H-And OPO2 -；R5It is hydrogen or-CR11R12-Y-Gb- Yc-[(CR9R10)y-O]k-R8Part, wherein：Each Y is independently selected from the following group, and the group is made up of the following：O, S, N-H or N-R8；And each R8Independently selected from the following group, the group is made up of the following：Alkyl, aryl and heteroaryl, the part is It is substituted or unsubstituted, and either replace or it is unsubstituted, the part is having less than 21 carbon；Each G is independent Ground is selected from the group, and the group is made up of the following：CO、SO2, SO, PO and PO2；R9And R10Independently selected from the following group, the group by with Lower every composition：H and C1-C4Alkyl；R11And R12Independently selected from the following group, the group is made up of the following：H and alkyl, or Carbonyl is may be combined to form when altogether；B=0 or 1；C can with=0 or 1, but if b=0, c it is necessary=0；Y be from 1 to 6 integer；K is the integer from 0 to 20；R6It is H, or alkyl, aryl or heteroaryl moieties；The part is substitution or not taken Generation；And X, if it exists, being suitable charge balancing counter ion, preferably works as R4When being hydrogen, X is present, is fitted The X of conjunction includes but is not limited to：Chloride, bromide, sulfate, methoxy sulfate (methosulphate), sulfonate, to first Benzene sulfonate, boron tetrafluoride and phosphate.
In in one aspect of the invention, bleaching catalyst has the structure corresponding to below general formula：
Wherein R13It is to include from three to 24 the branched alkyl of carbon atom (carbon atom for including branch) or comprising from one The individual straight chained alkyl to 24 carbon atoms；Preferably, R13Be include from eight to 18 the branched alkyl of carbon atom or comprising from Eight straight chained alkyls to 18 carbon atoms；Preferably, R13It is selected from the group, the group is made up of the following：2- propylheptyls, 2- butyl octyls, 2- pentylnonanyis, 2- hexyls decyl, n- dodecyl, n- myristyl, n- cetyl, n- 18 Alkyl, iso- nonyl, iso- decyl, iso- tritriacontyl and iso- pentadecyl；Preferably, R13It is selected from the group, the group is by the following Composition：2- butyl octyls, 2- pentylnonanyis, 2- hexyls decyl, iso- tritriacontyl and iso- pentadecyl.
Preferably, in addition to bleaching catalyst, particularly organic bleaching catalyst, bleaching component also includes source of peracid. Source of peracid can be selected from (a) pre-formed peracid；(b) percarbonate, perborate or persulfate (hydrogen peroxide source), preferably Combined with a kind of bleach-activating；Perhydrolase and ester (c), in textile or crust process step in water In the presence of be formed in situ peracid.
When it is present, based on said composition, peracid and/or bleach-activating generally with from 0.1wt% to 60wt%, from Amounts of the 0.5wt% to 40wt% or from 0.6wt% to 10wt% is present in composition.Can be by one or more hydrophobicity mistakes Acid or its precursor are used with one or more hydrophilic peracids or its combination of precursors.
The amount of hydrogen peroxide source and peracid or bleach-activating can be selected, to cause available oxygen (to come from peroxide Compound source) with the mol ratio of peracid it is from 1:1 to 35:1, or 2:1 to 10:1.
(6) bleaching catalyst-bleaching component can be provided by catalytic metal complex comprising metal.A type of bag Bleaching catalyst containing metal is following catalysis system, and the catalysis system includes the transition gold with the bleach catalyst activity limited Belong to cation (such as copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cation), with seldom or without the active auxiliary of bleach catalyst Metal cation (such as zinc or aluminium cations), and there is the stability limited for catalytic and complementary metal cation The spacer of constant, particularly ethylenediamine tetra-acetic acid, EDTMP and its water soluble salt.Such catalyst is draped over one's shoulders It is exposed in US 4430243.It is preferred that catalyst be described in WO 09/839406, US 6218351 and WO 00/012667.It is special You Xuanshi not transition-metal catalyst or therefore as the part of the multiple tooth N- donor ligands of cross-bridge.
If desired, composition in this can be catalyzed by manganese compound.Such compound and use level It is the catalyst well known and including being for example disclosed in US 5576282 based on manganese in the art.
Useful cobalt bleaching catalyst is known and is for example described in US 5597936 herein；In US 5595967. Such Co catalysts can easily be prepared by known program, the journey taught in such as US 5597936 and US 5595967 Sequence.
Composition in this can also compatibly include the transition metal complex of part, such as bipiperidine ketone (bispidone) (US 7501389) and/or macropolycyclic rigid ligand-be abbreviated as " MRL ".As a practical problem not It is used as limitation, composition in this and method can be adjusted, provides about at least about 100,000,000 points in Aqueous wash medium One of active MRL species, and will typically be in cleaning solution provide from 0.005ppm to 25ppm, from 0.05ppm to The 10ppm or MRL from 0.1ppm to 5ppm.
Transition metal bleach catalyst in suitable transition metal include such as manganese, iron and chromium.Suitable MRL Including 5,12- diethyl -1,5,8,12- 4-azabicyclos [6.6.2] hexadecane.Can easily it be prepared by known program suitable The program taught in the transition metal M RL of conjunction, such as US 6225464 and WO 00/32601.
(7) optical white-suitable optical white includes the Phthalocyanine Zinc, the aluminum phthalocyanine of sulfonation, xanthene dye of such as sulfonation And its mixture.Preferred bleaching component for being used in these compositions of the present invention includes hydrogen peroxide source, bleaching and lived Agent and/or organic peroxide acid, optionally by hydrogen peroxide source and bleach-activating and the combined reaction of bleaching catalyst And generation in situ.It is preferred that bleaching component include bleaching catalyst, preferably above-described organic bleaching catalyst.
Particularly preferred bleaching component is bleaching catalyst, particularly organic bleaching catalyst.
Exemplary bleaching system is also described in such as WO 2007/087258, WO 2007/087244, WO2007/087259 And in WO 2007/087242.
Fabric hueing agent- said composition can include fabric hueing agent.Suitable fabric hueing agent include dyestuff, dyestuff- Clay conjugates and pigment.Suitable dyestuff includes small molecule dyes and polymeric dye.Suitable small molecule dyes include The small molecule dyes being selected from the group, the group is made up of the following：Belong to the dyestuff of following color index (C.I.) classification：Directly Blue, direct red, direct purple, acid blue, acid red, acid violet, alkali blue, alkalescence purple and alkaline red or its mixture.
On the one hand, suitable small molecule dyes include the small molecule dyes being selected from the group, and the group is made up of the following： Color index (dyer and colourist association (S °C of iety of Dyers and Colorists), Bradford, Britain) is compiled Number directly purple 9, directly purple 35, directly purple 48, directly purple 51, directly purple 66, directly purple 99, directly indigo plant 1, directly indigo plant 71, directly Indigo plant 80, directly indigo plant 279, azogeramine 7, acid red 73, acid red 88, azogeramine 50, acid violet 15, acid violet 17, acid violet 24th, acid violet 43, acid red 52, acid violet 49, acid violet 50, Blue VRS 5, Blue VRS 7, acid blue 25, acid blue 29, acid Property indigo plant 40, acid blue 45, Acid Blue 75, acid blue 80, acid blue 83, acid blue 90 and Acid blue 113, acid black 1, alkalescence purple 1st, alkaline purple 3, alkalescence purple 4, alkaline purple 10, alkaline purple 35, alkali blue 3, alkali blue 16, alkali blue 22, alkali blue 47, alkali blue 66th, Blue 75, alkali blue 159 and its mixture.On the one hand, suitable small molecule dyes include the small molecule being selected from the group Dyestuff, the group is made up of the following：Color index (dyer and colourist association, Bradford, Britain) numbering acid violet 17th, acid violet 43, acid red 52, acid red 73, acid red 88, azogeramine 50, acid blue 25, acid blue 29, acid blue 45, Acid blue 113, directly acid black 1, indigo plant 1, directly indigo plant 71, directly purple 51 and its mixture.On the one hand, suitable small molecule dye Material includes the small molecule dyes being selected from the group, and the group is made up of the following：Color index (dyer and colourist association, Bradley Moral Ford, Britain) numbering acid violet 17, directly indigo plant 71, directly purple 51, directly indigo plant 1, acid red 88, azogeramine 50, acid blue 29th, Acid blue 113 or its mixture.
Suitable polymeric dye includes the polymeric dye being selected from the group, and the group is made up of the following：Comprising conjugated The polymer (dye-polymer conjugates) of chromogen and the polymer entered with chromogen combined polymerization in main polymer chain, and Its mixture.
On the one hand, suitable polymeric dye includes the polymeric dye being selected from the group, and the group is made up of the following： The substantive colouring agent of fabric under (Milliken (Milliken)) title, from least one chemically-reactive dyes With the dye-polymer conjugates for the polymer formation being selected from the group, the group is made up of the following：Comprising selecting free hydroxyl portion Point, one-level amine moiety, secondary amine part, thiol moiety and its mixture composition group part polymer.In still one side, Suitable polymeric dye includes the polymeric dye being selected from the group, and the group is made up of the following：Purple CT, Conjugated carboxymethyl cellulose (CMC) with reactive blue, reactive violet or active red dye, such as with C.I. reactive blues 19 (by plum lattice Ze Mu companies (Megazyme), Wicklow, Ireland, with ProductName AZO-CM-CELLULOSE, goes out under product code S-ACMC Sell) conjugated CMC, triphenyl-methane polymeric colorant of alkoxylate, the thiophene polymeric colorant of alkoxylate and Its mixture.
It is preferred that dope dye include findings that the brightening agent in WO 08/87497.These brightening agents can be by following knot Structure (I) is characterized：
Wherein R1And R2Can be independently selected from：
a)[(CH2CR’HO)x(CH2CR”HO)yH]
Wherein R ' is selected from the group, and the group is made up of the following：H、CH3、CH2O(CH2CH2O)zH and its mixture；Wherein R " is selected from the group, and the group is made up of the following：H、CH2O(CH2CH2O)zH and its mixture；Wherein x+y≤5；Wherein y >= 1；And wherein z=0 to 5；
b)R1=alkyl, aryl or aryl alkyl, and R2=[(CH2CR’HO)x(CH2CR”HO)yH]
Wherein R ' is selected from the group, and the group is made up of the following：H、CH3、CH2O(CH2CH2O)zH and its mixture；Wherein R " is selected from the group, and the group is made up of the following：H、CH2O(CH2CH2O)zH and its mixture；Wherein x+y≤10；Wherein y >= 1；And wherein z=0 to 5；
c)R1=[CH2CH2(OR3)CH2OR4] and R2=[CH2CH2(O R3)CH2O R4]
Wherein R3It is selected from the group, the group is made up of the following：H、(CH2CH2O)zH and its mixture；And wherein z= 0 to 10；
Wherein R4It is selected from the group, the group is made up of the following：(C1-C16) alkyl, aromatic yl group and its mixture；With And
D) wherein R1 and R2 can be independently selected from styrene oxide, glycidol methyl ether, isobutyl glycidyl ether, different Propyl glycidyl ether, tertiary butyl glycidyl ether, 2- hexyl glycidyl ethers and glycidol cetyl ether Amino addition compound product, is then the addition from 1 to 10 alkylene oxide unit.
The preferred whitening agent of the present invention can be characterized by following structure (II)：
Wherein R ' is selected from the group, and the group is made up of the following：H、CH3、CH2O(CH2CH2O)zH and its mixture；Wherein R " is selected from the group, and the group is made up of the following：H、CH2O(CH2CH2O)zH and its mixture；Wherein x+y≤5；Wherein y >= 1；And wherein z=0 to 5.
The other preferred brightening agent of the present invention can be characterized by following structure (III)：
Typically comprise the mixture with 5 EO groups altogether.Suitable preferred molecule is having in structure I Above " those of following sagging group in a " of part.
Table 1
R1 R2
R’ R” X y R’ R” x y
A H H 3 1 H H 0 1
B H H 2 1 H H 1 1
C=b H H 1 1 H H 2 1
D=a H H 0 1 H H 3 1
The other brightening agent used includes being described in that in US 2008/34511 (Uniliver (Unilever)) A bit.It is preferred that reagent be " purple 13 ".
Suitable dye clay conjugates includes the dye clay conjugates that is selected from the group, the group include at least one sun from Son/basic-dyeable fibre and terre verte and its mixture.On the one hand, suitable dye clay conjugates includes the dyestuff being selected from the group Clay conjugates, the group is made up of a kind of cation/basic-dyeable fibre and a kind of clay, and the cation/basic-dyeable fibre is selected from the group, The group is made up of the following：C.I. basic yellow 1 to 108, C.I. Basic Oranges 1 to 69, C.I. alkali red 1:1s to 118, C.I. are alkaline The alkaline brown 1 to 23 of purple 1 to 51, C.I. alkali blues 1 to 164, C.I. Viride Nitenses 1 to 14, C.I., CI basic blacks 1 to 11, and The clay is selected from the group, and the group is made up of the following：Montmorillonitic clay, hectorite clay, saponite clay and its mixture. Another aspect, suitable dye clay conjugates includes the dye clay conjugates being selected from the group, and the group is made up of the following： Montmorillonite alkali blue B7C.I.42595 conjugates, montmorillonite alkali blue B9C.I.52015 conjugates, montmorillonite alkalescence purple V3C.I.42555 conjugates, montmorillonite Viride Nitens G1C.I.42040 conjugates, the red R1C.I.45160 of montmorillonite alkalescence are conjugated Thing, the conjugates of montmorillonite C.I. basic blacks 2, hectorite alkali blue B7C.I.42595 conjugates, hectorite alkali blue B9C.I.52015 conjugates, hectorite alkalescence purple V3C.I.42555 conjugates, hectorite Viride Nitens G1C.I.42040 are conjugated Thing, the red R1C.I.45160 conjugates of hectorite alkalescence, the conjugates of hectorite C.I. basic blacks 2, saponite alkali blue B7C.I.42595 conjugates, saponite alkali blue B9C.I.52015 conjugates, saponite alkalescence purple V3C.I.42555 conjugates, soap Stone Viride Nitens G1C.I.42040 conjugates, the red R1C.I.45160 conjugates of saponite alkalescence, the conjugates of saponite C.I. basic blacks 2 and Its mixture.
Suitable pigment includes the pigment being selected from the group, and the group is made up of the following：Yellow scholar's ketone, indanthrone, comprising 1 to Chloride indanthrone, pyranthrone, dichloro pyranthrone, single bromine dichloro pyranthrone, Dibromo-dichloro pyranthrone, the tetrabromo skin of 4 chlorine atoms (wherein the imide group can be unsubstituted or by C1- for anthrone, perylene -3,4,9,10- tetracarboxylic acid diimides C3- alkyl or phenyls or heterocyclic group substitution, and wherein the phenyl and heterocyclic group can be additionally with not assigning Deliquescent substituent in water), anthrapyrimidine carboxylic acid amide, violanthrone, isoviolanthrone, triazine dioxin pigments, each molecule can It is chloro- with many bromines of the CuPc comprising up to 2 chlorine atoms, how chloro- CuPc or each molecule comprising up to 14 bromine atoms CuPc, and its mixture.
On the one hand, suitable pigment includes the pigment being selected from the group, and the group is made up of the following：Ultramarine (C.I. face Material is blue 29), ultramarine violet (C.I. pigment violet 1s 5) and its mixture.
Above-mentioned fabrics toner can be applied in combination (any mixture that can use fabric hueing agent).Suitable toning Agent is described in greater detail in US 7208459.The preferred levels of dyestuff in the present compositions be 0.00001wt% extremely 0.5wt% or 0.0001wt% to 0.25wt%.It is preferred that being used for the concentration of the dyestuff of processing and/or cleaning in water is From 1ppb to 5ppm, 10ppb to 5ppm or 20ppb to 5ppm.In preferred composition, the concentration of surfactant will be from 0.2 to 3g/l.
Capsule compound- said composition can include capsule compound.On the one hand, capsule compound includes a core, has Inner surface and the involucrum of outer surface, the involucrum encapsulate the core.
In the one side of the capsule compound, the core can include a kind of material being selected from the group, and the group is by following Items composition：Spices；Brightener；Dyestuff；Insect repellent；Silicone；Wax；Flavor enhancement；Vitamin；Fabric softener；Skin conditioner, On the one hand, it is paraffin；Enzyme；Antiseptic；Bleaching agent；Feelings agent (sensate)；And its mixture；And the involucrum can be wrapped A kind of material being selected from the group is included, the group is made up of the following：Polyethylene；Polyamide；Polyvinyl alcohol, optionally comprising other Comonomer；Polystyrene；Polyisoprene；Makrolon；Polyester；Polyacrylate；Aminoplast, it is on the one hand, described Aminoplast can include polyureas, polyurethane and/or polyurea polyurethanes, on the one hand, and it is sub- that the polyureas can include polyoxy MU and/or melamino-formaldehyde；Polyolefin；Polysaccharide, on the one hand, the polysaccharide can include alginate and/or shell Glycan；Gelatin；Shellac；Epoxy resin；Polyvinyl water insoluble inorganic substance；Silicone；And its mixture.
In the one side of the capsule compound, the core can include spices.
In the one side of the capsule compound, the involucrum can include melamino-formaldehyde and/or the melamine of crosslinking Amine formaldehyde.
On the one hand, core material and involucrum can be included by disclosing suitable capsule compound, and the involucrum is at least partly Ground surrounds the core material.At least 75%, 85% or 90% of the capsule compound can have from 0.2MPa to 10MPa, Fracture strength from 0.4MPa to 5MPa, from 0.6MPa to 3.5MPa or from 0.7MPa to 3MPa；And with from 0 to 30%, Revealed from 0 to 20% or from 0 to 5% beneficial agent.
On the one hand, at least 75%, 85% or 90% of the capsule compound can have from 1 to 80 micron, from 5 to 60 Micron, from 10 to 50 microns or from 15 to 40 microns of granularity.
On the one hand, at least 75%, 85% or 90% of the capsule compound can have from 30 to 250nm, from 80 to 180nm or the particle wall thickness from 100 to 160nm.
On the one hand, the core material of the capsule compound can include a kind of selected from the group being made up of spices raw material Material, and/or optionally include a kind of material being selected from the group, the group is made up of the following：Vegetable oil, including pure plant Oil and/or blending vegetable oil, including castor oil (caster oil), coconut oil, cottonseed oil, grape-kernel oil, rapeseed, soybean Oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm-kernel oil, castor oil (castor Oil), lemon oil and its mixture；The ester of vegetable oil, ester, including dibutyl adipate, Dibutyl phthalate, adipic acid butyl benzyl Ester, adipic acid octyl group benzyl ester, tricresyl phosphate, trioctyl phosphate and its mixture；Straight or branched hydrocarbon, including with higher than Those straight or branched hydrocarbon of about 80 DEG C of boiling point；Partially hydrogenated terphenyl, dialkyl phthalate, alkyl biphenyl (including single isopropyl biphenyl), naphthalene (including dipropyl naphthalene), benzin (including kerosene), mineral oil and its mixing of alkylation Thing；Aromatic solvent, including benzene, toluene and its mixture；Silicone oil；And its mixture.
On the one hand, the wall material of the capsule compound can include suitable resin, and the resin includes the anti-of aldehyde and amine Product is answered, suitable aldehyde includes formaldehyde.Suitable amine includes melamine, urea, benzoguanamine, glycoluril and its mixture.It is adapted to Melamine include melamine methylol, the melamine methylol methylated, imino group melamine and its mixture. Suitable urea includes dimethylol urea, the dimethylol urea methylated, urea-resorcinol and its mixture.
On the one hand, before, during or after capsule compound is added into composition, suitable formaldehyde scavenger can be with It is used together with the capsule compound in for instance in capsule slurry and/or added in such composition.Suitable capsule can To pass through US 2008/0305982；And/or the following of US 2009/0247449 is taught to be made.
In a preferred aspect, said composition can also include deposition acid, be preferably made up of the following group, and the group includes cation Or non-ionic polymers.Suitable polymer includes cationic starch, cationic hydroxy ethyl cellulose, polyvinyl formal, Chinese scholartree Bean gum, mannosan, xyloglucan, tamarind gum, polyethyleneterephthalate are set, and includes dimethylaminoethyl acrylate methyl The polymer of amine ethyl ester, the monomer being optionally selected from the group with one or more, group includes acrylic acid and acrylamide.
Spices- on the one hand, said composition includes the spices containing the one or more spices raw material being selected from the group, should Group is made up of the following：Double -2- the propyl alcohol of 1,1 '-epoxide；Isosorbide-5-Nitrae-cyclohexane dicarboxylic acid, diethylester；((ethoxymethyl) epoxide) ring Dodecane；1,3- nonanediol, monoacetate；(3- methylbutoxy groups) acetoacetate, 2- propylene base esters；Beta-methyl cyclododecane second Alcohol；2- methyl -3- [(bicyclic [2.2.1] the hept- 2- yls of 1,7,7- trimethyls) oxygen] -1- propyl alcohol；Omega-pentadecanolide；Alpha-Methyl-benzene first Alcohol ester；Trans -3- ethyoxyls -1,1,5- trimethyl-cyclohexanes；4- (1,1- dimethyl ethyls) adnoral acetate；Ten dihydros- 3a, 6,6,9a- tetramethyl naphtho- [2,1-b] furans；Beta-methyl benzenpropanal；Beta-methyl -3- (1- Methylethyls) benzenpropanal；4- benzene Base -2- butanone；2- methyl butyric acid, ethyl ester；Benzaldehyde；2- methyl butyric acid, 1- Methylethyls；Dihydro -5- amyl groups -2 (3H) furanone；(2E)-1- (2,6,6- trimethyl-2- cyclohexene-1- bases)-2- butene-1 -one；Lauric aldehyde；The hendecanal；2- second Base-α, alpha-alpha-dimethyl benzenpropanal；Capraldehyde；α, alpha-alpha-dimethyl benzyl carbinol acetic acid esters；2- (phenylmethylene) octanal；2-[[3-[4- (1,1- dimethyl ethyl) phenyl] -2- methyl propylenes] amino] benzoic acid, methyl ester；1- (2,6,6- trimethyl -3- hexamethylenes Alkene-1- bases)-2- butene-1 -one；2- amyl group cyclopentanone；3- oxo -2- amyl group cyclopentaneacetic acids, methyl ester；4- hydroxyl -3- methoxies Benzaldehyde；Vanirom；Alismone；1- (4- aminomethyl phenyls) ethyl ketone；(3E)-4-(2,6,6- Trimethyl-1- cyclohexene-1- bases)-3- butene-2 -one；(3E) -4- (2,6,6- trimethyl -2- cyclohexene -1- bases) -3- butylene - 2- ketone；Benzyl carbinol；2H-1- chromen-2-ones；4-methoxybenzaldehyde；10- undecylene aldehydes；Propionic acid, phenyl methyl ester；β- Methyl fenipentol；1,1- diethoxy -3,7- dimethyl -2,6- octadienes；α, alpha-alpha-dimethyl benzyl carbinol；(2E)-1-(2,6,6- Trimethyl-1- cyclohexene-1- bases)-2- butene-1 -one；Acetic acid, phenyl methyl ester；Cyclohexylpropionic acid, 2- propylene base esters；Caproic acid, 2- propylene base esters；1,2- dimethoxy-4 's-(2- acrylic) benzene；The ring of 1,5- dimethyl-two [3.2.1] octane -8- ketoximes；4- (4- hydroxy-4-methyls amyl group) -3- cyclohexene-1-formaldehydes；3- butene-2 -ol；2- [[[2,4 (or 3,5)-dimethyl -3- hexamethylenes Alkene -1- bases] methylene] amino] benzoic acid, methyl ester；8- ring hexadecene-1 -one；Methylionone；2,6- dimethyl -7- Octen-2-ol；2- methoxyl groups -4- (2- acrylic) phenol；(2E) -3,7- dimethyl -2,6- octadiene -1- alcohol；2- hydroxyls-benzene Formic acid, (3Z) -3- hexene esters；2- tridecylene nitriles；4- (2,2- dimethyl -6- methylenecyclohexyls) -3- methyl -3- butylene - 2- ketone；Tetrahydrochysene -4- methyl -2- (2- methyl-1-propylenes base) -2H- pyrans；Acetoacetate, (2- methylbutoxy groups)-, 2- propylene Base ester；Benzoic acid, 2- hydroxyls-, 3- methyl butyl esters；2- butene-1 -one, 1- (2,6,6- trimethyl-1- cyclohexene-1- bases)-, (Z)-；Ring valeric acid, 2- hexyl -3- oxos -, methyl ester；Benzenpropanal, 4- ethyl-α, α .- dimethyl-；3- cyclohexene -1- pyrroles Cough up formaldehyde, 3- (4- hydroxy-4-methyls amyl group)-；Ethyl ketone, 1- (2,3,4,7,8,8a- hexahydros -3,6,8,8- tetramethyl -1H-3a, 7- methylene bridge Azulene -5- bases) -, [3R- (3. α, 3a. β, 7. β, 8a. α)] -；The hendecanal, 2- methyl -2H- pyran-2-ones, 6- fourths tetrahydro-；Benzenpropanal, 4- (1,1- dimethyl ethyl)-α .- methyl-；2 (3H)-furanones, 5- dihydros in heptan-；Benzene first Acid, 2- [(7- hydroxyl -3,7- dimethyl octamethylene) amino] -, methyl；Benzoic acid, 2- hydroxyls-, phenyl methyl ester；Naphthalene, 2- first Epoxide-；2- cyclopentene-1-ones, 2- hexyls-；2 (3H)-furanones, 5- dihydros in heptan-；Oxirane carboxylic acid, 3- methyl -3- benzene Base-, ethyl ester；The ring of 2- oxygen two [2.2.2] octane, 1,3,3- trimethyl-；Fenipentol, γ .- methyl-；3- octanols, 3,7- bis- Methyl-；3,7- dimethyl -2,6- octadiene nitriles；3,7- dimethyl -6- octen-1-ols；Terpin alcohol acetic ester；2- methyl -6- is sub- Methyl -7- octen-2-ols, dihydro derivative；3a, 4,5,6,7,7a- hexahydro -4,7- methylene bridge -1H- indenes -6- alcohol propionic esters； 3-M2BOL acetic acid esters；(Z)-blatter alcohol acetic acid esters；2- ethyls -4- (2,2,3- trimethyl -3- rings penta Alkene -1- bases) -2- butene-1-ols；4- (octahydro -4,7- methylene bridge -5H- indenes -5- subunits)-butyraldehyde；3-2,4- dimethyl-ring Hexene -1- pyrrole aldehydes；1- (1,2,3,4,5,6,7,8- octahydro -2,3,8,8- tetramethyl -2- naphthalenes)-ethyl ketone；2- hydroxyls-benzene first Acid, methyl ester；2- hydroxy-benzoic acids, hexyl ester；2- phenoxy-ethanols；2- hydroxy-benzoic acids, amyl group ester；2,3- heptadione； 2- hexen-1-ols；6- octen-2-ols, 2,6- dimethyl-；Damascenone (α, beta, gamma or δ and its mixture), 4,7- methylene bridges- 1H- indenes -6- alcohol, 3a, 4,5,6,7,7a- hexahydros -, acetic acid esters；9- undecylene aldehydes；8- undecylene aldehydes；Isocyclocitral；Ethyl ketone, 1- (1,2,3,5,6,7,8,8a- octahydro -2,3,8,8- tetramethyl -2- naphthalenes) -；3- cyclohexene -1- pyrrole aldehydes, 3,5- diformazans Base-；3- cyclohexene -1- pyrrole aldehydes, 2,4- dimethyl -；1,6- octadiene -3- alcohol, 3,7- dimethyl -；1,6- octadienes -3- Alcohol, 3,7- dimethyl-, acetic acid esters；Lilial (p-t-Bucinal), and cyclopentanone, 2- [2- (4- methyl -3- cyclohexene -1- Base) propyl group]-and 1- methyl -4- (1- methyl ethylenes) cyclohexene and its mixture.
On the one hand, said composition can include encapsulated perfume particle, and the particle includes water soluble hydroxy compound Or carbamide or the polyvinyl alcohol of modification.On the one hand, capsule compound includes the water-soluble solid-based of (a) at least part Matter, includes one or more water soluble hydroxy compounds, preferred starch；And the perfumery oil that (b) is encapsulated by the solid matrix.
On the other hand, the spices can be pre-composite with polyamines (preferably polyethyleneimine), to form schiff bases (Schiff base)。
Polymer- said composition can include one or more polymer.Example be carboxymethyl cellulose, it is poly- (vinyl- Pyrrolidones), PEG, poly- (vinyl alcohol), poly- (vinylpyridine-N-oxide), poly- (vinyl imidazole), polycarboxylic acids Ester (such as polyacrylate), maleic acid/acrylic copolymer and lauryl methacrylate/acrylic copolymer.
Said composition can include one or more amphiphilic cleaning polymer, such as chemical combination with following general structure Thing：Double ((C2H5O)(C2H4O)n)(CH3)-N+-CxH2x-N+-(CH3)-bis- ((C2H5O)(C2H4O) n), wherein n=is from 20 to 30, And x=is from 3 to 8, or its sulphation or sulfonation variant.
Said composition can include amphiphilic alkoxylate grease cleaning polymer, these polymer have balance it is hydrophilic and Hydrophobic property so that they remove fat particles from fabric and surface.The amphiphilic alkoxylate grease cleaning polymer of the present invention Specific embodiment include a core texture and multiple Alkoxylated groups for being connected with that core texture.These can be wrapped The poly- olefin(e) imines (polyalkylenimine) of alkoxylate is included, preferably with interior poly-ethylene oxide block and outer polycyclic oxygen Propane block.
Here, the polycarboxylate (such as those prepared from polyacrylate) of alkoxylate can be used for providing other oil Fat removal capacity.Such material is described in WO 91/08281 and PCT 90/01815.Chemical Shangdi, these materials include poly- Acrylate, has an ethoxy side chain per 7-8 acrylate unit.Side chain has chemical formula-(CH2CH2O)m(CH2)nCH3, wherein m is 2-3 and n is 6-12.Side chain is that ester is connected to polyacrylate " main chain ", to provide " comb shape " polymer Type structure.Molecular weight can be different, but are typically in the range of 2000 to 50,000.The polycarboxylic acids of such alkoxylate Ester can be including composition in this from 0.05wt% to 10wt%.
The present invention isoprenoid derived from surfactant, and with other cosurfactants and other adjuvants The mixture that composition is formed together is particularly suitable for using with amphipathic graft copolymer, and preferably the amphipathic graft copolymer includes (i) polyethylene glycol backbone；And (ii) and at least one hanging portion, selected from polyvinyl acetate, polyvinyl alcohol and its mixing Thing.It is preferred that amphipathic graft copolymer be by BASF (BASF) supply Sokalan HP22.Suitable polymer include with The polyethylene oxide copolymer of machine graft copolymer, preferably polyvinyl acetate grafting, with PEO main chain and multiple Polyvinyl acetate ester side chain.The molecular weight of PEO main chain is preferably 6000, and PEO and polyvinyl acetate The weight ratio of ester is 40 to 60, and every 50 ethylene oxide unit(s)s have not more than 1 grafting site.
Carboxylate polymerThe composition of-the present invention also includes one or more carboxylate polymers, for example maleate/ Acrylate randomcopolymer or polyacrylate homopolymers.On the one hand, the carboxylate polymer is that have from 4,000Da extremely 9,000Da or molecular weight from 6,000Da to 9,000Da polyacrylate homopolymers.
Soil release polymerThe composition of-the present invention can also include one or more soil release polymers, these polymer tool Just like the structure as defined in one of following structure (I), (II) or (III)：
(I)-[(°CHR1-CHR2)a-O-°C-Ar-CO-]d
(II)-[(°CHR3-CHR4)b-O-°C-sAr-CO-]e
(III)-[(°CHR5-CHR6)c-OR7]f
Wherein：
A, b and c are from 1 to 200；
D, e and f are from 1 to 50；
Ar is the phenylene of 1,4- substitutions；
SAr is the phenylene of 1,3- substitutions, and the phenylene is on 5 by SO3Me replaces；
Me is Li, K, Mg/2, Ca/2, Al/3, ammonium, single-, two-, three-or tetra-allkylammonium, and wherein alkyl is C1-C18Alkyl Or C2-C10Hydroxy alkyl, or its mixture；
R1、R2、R3、R4、R5And R6Independently selected from H or C1-C18N- or iso- alkyl；And
R7It is the C of straight or branched1-C18Alkyl, or straight or branched C2-C30Alkenyl, or with 5 to 9 carbon atoms Cycloalkyl, or C8-C30Aryl, or C6-C30Aryl alkyl.
Suitable soil release polymer is polyester soil release polymers, such as Repel-o-tex polymer, including Repel-o- Tex, SF-2 and SRP6, are supplied by Luo Diya (Rhodia).Other suitable soil release polymers include Texcare polymer, bag Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325 are included, by Clariant (Clariant) supply.Other suitable soil release polymers are Marloquest polymer, for example Marloquest SL, by Sa Sol (Sasol) is supplied.
Cellulosic polymerThe composition of-the present invention also includes one or more cellulosic polymers, including selected from alkyl Those of cellulose, alkyl alkoxy alkylcellulose, carboxyalkyl cellulose, alkylcarboxyalkyl cellulose.On the one hand, Cellulosic polymer is selected from the group, and the group includes carboxymethyl cellulose, methylcellulose, methyl hydroxyl ethyl cellulose, methyl Carboxymethyl cellulose and its mixture.On the one hand, carboxymethyl cellulose have from 0.5 to 0.9 degree of substitution by carboxymethyl and Molecular weight from 100,000Da to 300,000Da.
Enzyme-said composition can include the one or more enzymes for providing clean-up performance and/or fabric care benefits.Suitable The example of enzyme includes but is not limited to hemicellulase, peroxidase, protease, cellulase, zytase, lipase, phosphorus Lipase, esterase, cutinase, pectase, mannonase pectin lyase, keratinase, reductase, oxidizing ferment, phenol oxidation Enzyme, lipoxygenase, lignoenzyme, amylopectase, tannase, poly-pentose enzyme, horse traction receive enzyme (malanase), beta glucan Enzyme, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase or its mixture.Typical group Conjunction is enzymatic mixture, can be comprising such as protease and lipase together with amylase.When present in the composition, it is foregoing in addition Enzyme can based on the weight of composition with from 0.00001wt% to 2wt%, from 0.0001wt% to 1wt% or from The level of 0.001wt% to 0.5wt% zymoproteins is present.
In general, one or more selected enzyme viabilities should (that is, optimal pH, with other compatible with selected detergent Compatibility of enzyme and non-enzyme component, etc.), and one or more enzymes should exist with effective dose.
On the one hand, enzyme preferably will include a kind of cellulase.Suitable cellulase includes bacterium or originated from fungus Those.Mutant or protein engineered mutant including chemical modification.Suitable cellulase is included from following The cellulase of category：Bacillus, pseudomonas, Humicola, Fusarium, Thielavia, acremonium, for example Disclosed in US 4435307, US 5648263, US 5691178, US 5776757 and WO 89/09259 by special detritus The fungal cellulase that mould, thermophilic fungus destroyed wire and Fusarium oxysporum are produced.
Particularly suitable cellulase is alkalescence or neutral cellulase with Color care benefit.Such cellulose The example of enzyme is EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, the fibre described in WO 98/08940 The plain enzyme of dimension.Other examples are cellulase variants, such as WO 94/07998, EP 0531315, US 5457046, US 5686593, Those described in US 5763254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
Commercially available cellulase includes CelluzymeTMAnd CarezymeTM(Novozymes Company (Novozymes A/ S))、ClazinaseTMAnd Puradax HATM(international corporation of Jie Neng sections (Genencor International )) and KAC-500 (B) Inc.TM(Kao Corp (Kao Corporation)).
On the one hand, enzyme preferably will include protease.Suitable protease includes bacterium, fungi, plant, viral or dynamic Those of thing source, such as plant or microbe-derived.Preferred microorganism is originated.Mutant or protein including chemical modification The mutant of engineering.It can be alkali protease, such as serine protease or metalloproteinases.Serine protease can To be, for example, S1 families (such as trypsase) or S8 families (such as subtilopeptidase A).Metalloproteinases, which may, for example, be, to be come from Such as family M4 thermolysin or other metalloproteinases, such as those from M5, M7 or M8 family.
Term " novel subtilases " refers to according to Si Aisen (Siezen) et al., protein engineering (Protein Engng.) 4 (1991) 719-737 and Si Aisen et al., (1997) 501-523 of protein science (Protein Science) 6 serine Protease subgroup.Serine protease is to be characterized as thering is the egg with the serine of substrate formation covalent adduct in avtive spot One subgroup of white enzyme.Novel subtilases can be divided into 6 sub-portions, i.e. subtilopeptidase A family, thermophilic protease man Race, Proteinase K family, lantibiotic peptase (Lantibiotic peptidase) family, Kexin families and Pyrolysin families.
The example of novel subtilases is derived from those of bacillus, for example, be described in US 7262042 and WO09/ Bacillus lentus, Alkaliphilic bacillus in 021867, bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus And bacillus gibsonii；With subtilopeptidase A slow (lentus), the bacillus subtilis protein being described in WO 89/06279 Enzyme promise and (Novo), subtilopeptidase A Carlsberg (Carlsberg), bacillus licheniformis, subtilopeptidase A BPN ', Subtilopeptidase A 309, subtilopeptidase A 147 and subtilopeptidase A 168 and it is described in (WO 93/18140) In protease P D138.Other useful protease can be described in WO 92/175177, WO 01/016285, WO 02/ Those in 026024 and WO 02/016547.The example of trypsin like proteases is that (such as pig or ox come trypsase Source) and Fusarium protease (being described in WO 89/06270, WO 94/25583 and WO 05/040372), and derive from The chymotrypsin (being described in WO 05/052161 and WO 05/052146) of cellulomonas cartae (Cellumonas).
Further preferred protease is the alkali protease from bacillus lentus DSM 5483 (such as in such as WO Described in 95/23221) and its variant (in WO 92/21760, WO 95/23221, EP 1921147 and EP 1921148 Description).
The example of metalloproteinases is as being described in WO 07/044993 (international corporation of Jie Neng sections (Genencor Int.)) In metalloprotease, for example from bacillus amyloliquefaciens those.
The example of useful protease is the variant being described in the following：WO 92/19729、WO 96/034946、 WO 98/20115、WO 98/20116、WO 99/011768、WO 01/44452、WO 03/006602、WO 04/03186、WO 04/041979th, WO 07/006305, WO 11/036263, WO 11/036264, especially in the one or more of following position In have substitution variant：3、4、9、15、27、36、57、68、76、87、95、96、97、98、99、100、101、102、103、 104、106、118、120、123、128、129、130、160、167、170、194、195、199、205、206、217、218、222、 224th, 232,235,236,245,248,252 and 274, use BPN ' to be numbered.It is highly preferred that these novel subtilases become Body can include following mutation：S3T、V4I、S9R、A15T、K27R、*36D、V68A、N76D、N87S,R、*97E、A98S、S99G, D,A、S99AD、S101G,M,R、S103A、V104I,Y,N、S106A、G118V,R、H120D,N、N123S、S128L、P129Q、 S130A、G160D、Y167A、R170S、A194P、G195E、V199M、V205I、L217D、N218D、M222S、A232V、 K235L, Q236H, Q245R, N252K, T274A (use BPN ' to be numbered).
Suitable commercially available protease is included with trade nameDuralaseTm、DurazymTm、Ultra、Ultra、 Ultra、Ultra、AndThose sold, it is all these can be withOr(Novi Letter company (Novozymes A/S)) sell；Those sold with following trade name： PreferenzTm、PurafectPurafect PurafectEffectenzTm、 And(Danisco/E.I.Du Pont Company (Danisco/DuPont)), AxapemTM(Ji Sitebu Luo Kadesi companies (Gist-Br °C of ases N.V.)), BLAP (sequence is shown in US 5352604 Figure 29) and its variant (Chinese High share (Henkel AG)) and KAP (Alkaliphilic bacillus subtilopeptidase A) from Kao Corp (Kao).
On the one hand, enzyme preferably will include a kind of amylase.Suitable amylase can be that alpha-amylase or glucose form sediment Powder enzyme and can be bacterium or originated from fungus.Mutant or protein engineered mutant including chemical modification.Form sediment Powder enzyme includes for example being derived from the alpha-amylase of bacillus, such as GB 1296839 lichens gemma bar in greater detail The alpha-amylase of the specific strain of bacterium.
Suitable amylase is included with the SEQ ID NO in WO 95/10603:3 amylase or itself and SEQ ID NO:3 have the variant of 90% sequence identity.It is preferred that variant be described in WO 94/02597, WO 94/18314, WO 97/ 43424 and WO 99/019467 SEQ ID NO:In 4, for example, there is the change of substitution at one or more of following position place Body：15、23、105、106、124、128、133、154、156、178、179、181、188、190、197、201、202、207、208、 209th, 211,243,264,304,305,391,408 and 444.
Different suitable amylase is included with the SEQ ID NO in WO 02/010355:6 amylase or its with SEQ ID NO:6 have the variant of 90% sequence identity.SEQ ID NO:6 preferred variants are that have in position 181 and 182 There is missing and there are those replaced in position 193.
Other suitable amylase are to include being shown in WO 2006/066594 SEQ ID NO:Solution starch is derived from 6 The residue 1-33 of the alpha-amylase of the bacillus and SEQ ID NO for being shown in WO 2006/066594:Bacillus licheniformis in 4 The residue 36-483 of alpha-amylase hybrid alpha-amylases or its variant with 90% sequence identity.This heterozygosis alphalise starch The preferred variants of enzyme are those in one or more of following position with substitution, missing or insertion：G48、T49、 G107, H156, A181, N190, M197, I201, A209 and Q264.SEQ ID NO including being shown in WO 2006/066594:6 In the alpha-amylase from bacillus amyloliquefaciens residue 1-33 and SEQ ID NO:4 residue 36-483 heterozygosis α- The most preferably variant of amylase is with following substituted those：
M197T；
H156Y+A181T+N190F+A209V+Q264S；Or
G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S。
Suitable other amylase is with the SEQ ID NO in WO 99/019467:6 amylase or itself and SEQ ID NO:6 have the variant of 90% sequence identity.SEQ ID NO:6 preferred variants are one or many in following position There are those of substitution, missing or insertion in individual：R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylase is those in position R181 and G182 or position H183 and G184 with missing.
The other amylase that can be used is the SEQ ID NO with WO 96/023873:1、SEQ ID NO:3、SEQ ID NO:2 or SEQ ID NO:7 those or itself and SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:7 have the variant of 90% sequence identity.SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 or SEQ ID NO:7 Preferred variants be in one or more of following position have substitution, missing or insertion those：140、181、182、 183rd, 184,195,206,212,243,260,269,304 and 476.Preferred variant is in position 181 and 182 or position There are those of missing in 183 and 184.SEQ ID NO:1、SEQ ID NO:2 or SEQ ID NO:7 most preferred amylase Variant is that have missing and one in position 140,195,206,243,260,304 and 476 in position 183 and 184 Or it is multiple in have substitution those.
Other amylase that can be used are with the SEQ ID NO in WO 08/153815:2nd, in WO 01/66712 SEQ ID NO:10 amylase or its SEQ ID NO with WO 08/153815:2 have 90% sequence identity or and WO SEQ ID NO in 01/66712:10 have the variant of 90% sequence identity.SEQ ID NO in WO 01/66712:10 Preferred variants be in one or more of following position have substitution, missing or insertion those：176、177、178、 179th, 190,201,207,211 and 264.
Other suitable amylase is with the SEQ ID NO in WO 09/061380:2 amylase or itself and SEQ ID NO:2 have the variant of 90% sequence identity.SEQ ID NO:2 preferred variants are one or many in following position Those of truncation and/or substitution with C-terminal, missing or insertion in individual：Q87、Q98、S125、N128、T131、T165、 K178、R180、S181、T182、G183、M201、F202、N225、S243、N272、N282、Y305、R309、D319、Q320、 Q359, K444 and G475.SEQ ID NO:2 more preferably variant is that have substitution at one or more of following position place Those：Q87E,R、Q98R、S125A、N128C、T131I、T165I、K178L、T182G、M201L、F202Y、N225E,R、 N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E and G475K, and/or in position R180 and/or S181 missing or the missing in position T182 and/or G183.SEQ ID NO:2 most preferred amylase variant be have with Those lower substituted：
N128C+K178L+T182G+Y305R+G475K；
N128C+K178L+T182G+F202Y+Y305R+D319T+G475K；
S125A+N128C+K178L+T182G+Y305R+G475K；Or
S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are C- ends Truncate and optionally further at position 243 include substitution and/or at position 180 and/or position 181 include lack Lose.
Other suitable amylase are with the SEQ ID NO in WO 01/66712:12 alpha-amylase or with SEQ ID NO:12 have the variant of at least 90% sequence identity.It is preferred that amylase variant be the SEQ ID in WO 01/66712 NO:12 following position it is one or more in have substitution, missing or insertion those：R28、R118、N174；R181、 G182、D183、G184、G186、W189、N195、M202、Y298、N299、K302、S303、N306、R310、N314；R320、 H324、E345、Y396、R400、W439、R444、N445、K446、Q449、R458、N471、N484.Particularly preferred amylase Including being lacked with D183 and G184 and with substitution R118K, N195F, R320K and R458K variant, and in addition in choosing There is the variant of substitution from one or more positions of the following group：M9、G149、G182、G186、M202、T257、Y295、N299、 M323, E345 and A339, the variant most preferably in all these positions in addition with substitution.
Other examples are amylase variants, such as in WO2011/098531, WO2013/001078 and WO2013/ Those described in 001087.
Commercially available amylase is DuramylTM、TermamylTM、Termamyl UltraTM、FungamylTM、BanTM、 StainzymeTM、Stainzyme PlusTM、SupramylTM、NatalaseTM, Liquozyme X and BANTM (coming from Novozymes Company (Novozymes A/S)),(in 9000Biozym Biotech Trading GmbH, Wehlistrasse 27b, A-1200 Vienna, Austria) and RapidaseTM、PurastarTM/EffectenzTM、 Powerase、Preferenz S100、Preferenx S110、OPTISIZE HTAnd PURASTAR(Danisco/E.I.Du Pont Company (Danisco/DuPont)) and(Kao Corp (Kao))。
Suitable lipase and cutinase include those of bacterium or originated from fungus.Including chemical modification or protein work The mutant enzyme of journey.Example includes the lipase belonged to from thermophilic fungal, for example, be such as described in EP 258068 and EP Thermomyces lanuginosus (being named as thin cotton like humicola lanuginosa previously) is come from 305216；Cutinase from Humicola, Such as Humicola insolens (WO 96/13580)；(some in these rename the lipase of bacterial strain from pseudomonas now For primary gram of Hall Bordetella), such as Pseudomonas alcaligenes or pseudomonas pseudoalcaligenes (EP218272), Pseudomonas cepacia (EP 331376), pseudomonas strain SD705 (WO 95/06720＆WO96/27002), Wisconsin pseudomonad (P.wisconsinensis)(WO 96/12012)；GDSL- type streptomyces lipase (WO 10/065455)；From rice blast The cutinase (WO 10/107560) of germ；Cutinase (US 5,389,536) from pseudomonas mendocina；From brown The thermophilic lipase (WO 11/084412, WO 13/033318) for splitting spore bacterium (Thermobifida fusca)；Stearothermophilus soil Bacillus lipase (WO 11/084417)；Lipase (WO 11/084599) from bacillus subtilis；And come from Streptomyces griseus (WO 11/150157) and the lipase (WO 12/ of rotation streptomycete (S.pristinaespiralis) 137147)。
Other examples are lipase Variants, for example, be described in EP 407225, WO 92/05249, WO 94/01541, WO 94/25578、WO 95/14783、WO 95/30744、WO 95/35381、WO 95/22615、WO96/00292、WO 97/ 04079th, WO 97/07202, WO 00/34450, WO 00/60063, WO 01/92502, WO 07/87508 and WO 09/ Those in 109500.
It is preferred that commercialization lipase product include LipolaseTM、LipexTM、Lipex EvityTM、Lipex 105TTM、 LipolexTMWith Lip °C of leanTM(Novozymes Company), Lumafast (come from Genencor Company (Genencor)) and Lipomax (comes from Ji Site-Bock De Si companies (Gist-Br °C of ades)).
Other examples are the lipase of sometimes referred to as acyltransferase or Perhydrolase again, such as with antarctic candida (Candida antarctica) lipase A has the acyltransferase (WO 10/111143) of homology, from the dirty branch of shame Acyltransferase (WO 05/56782), the Perhydrolase from the families of CE 7 of bacillus (Mycobacterium smegmatis) The variant of (WO 09/67279) and M. smegmatis perhydrolase (steps the limited public affairs of textile dyeization especially from Hensel Take charge of S54V used in the commercial product Gentle Power Bleach of (Huntsman Textile Effects Pte Ltd) Variant) (WO 10/100028).
On the one hand, what other preferred enzymes included microbial origin shows inscribe-β-Isosorbide-5-Nitrae-dextranase activity Endoglucanase (EC3.2.1.4), including (polypeptide has endogenous bacterial peptide for the member of bacillus With the amino acid sequence SEQ ID NO in US 7141403:The sequence of the uniformity of 2 at least 90%, 94%, 97% or 99%) with And its mixture.Suitable endoglucanase is in trade (brand) nameWith(Novozymes Company) Lower sale.
Other preferred enzymes are included in trade (brand) nameThe pectin of lower sale Lyases, and in trade (brand) name(Novozymes Company) and(Danisco/E.I.Du Pont Company (Danisco/DuPont) mannase sold under).
This one or more detergent enzyme can include the independent additive of one or more enzymes by addition, or by adding Plus combined additive including all these enzymes and be included in detergent composition.The detergent additives of the present invention, i.e., Independent additive or combined additive, can be configured to, such as particle, liquid, slurry.It is preferred that detergent additives match somebody with somebody Product is particle, especially non-pulverizing particle；Liquid, especially stabilized liquid；Or slurry.
Non-pulverizing particle can be manufactured for example as disclosed in US 4106991 and US 4661452, and can be optional Ground is coated with by method as known in the art.The example of waxy coating materials is with 1000 to 20000 molar average Poly- (oxirane) product (polyethylene glycol, PEG) of weight；With the ethoxyquin nonyl benzene from 16 to 50 ethylene oxide units Phenol；B oxidation fat alcohol, the wherein alcohol contain 12 to 20 carbon atoms, and wherein have 15 to 80 ethylene oxide units； Fatty alcohol；Aliphatic acid；And the monoglyceride and diglyceride and triglycerides of aliphatic acid.Suitable for passing through fluid bed skill The example of the film-forming coating materials of art application is provided in GB 1483591.Liquid enzyme formulation can have been established for example by basis Method addition polyalcohol (such as propane diols), sugar or sugar alcohol, lactic acid or boric acid stablizes.Shielded enzyme can be according to EP It is prepared by the method that is disclosed in 238216.
Dye transfer inhibitorThe composition of-the present invention can also include one or more dye transfer inhibitors.It is adapted to Polymeric dye transfer inhibitor include but is not limited to polyvinyl pyrrolidone polymers, polyamines N- oxide polymers, N- The copolymer, Ju Yi Xi oxazolidones and polyvinyl imidazole or its mixture of vinylpyrrolidone and N- vinyl imidazoles.When When being present in composition, dye transfer inhibitor can by from 0.0001wt% to 10wt%, from 0.01wt% to 5wt% or Level from 0.1wt% to 3wt% is present.
BrightenerThe composition of-the present invention can also include other component that can be to the color goods cleaned, example Such as brightener.
Said composition can include C.I. brighteners 260, in the alpha-crystal form with following structure：
On the one hand, brightener is cold water soluble brightener, for instance in the C.I. brighteners of alpha-crystal form 260.On the one hand, brightener is mostly in alpha-crystal form, it means that typically at least 50wt%, at least 75wt%, extremely Few 90wt%, at least 99wt% or even substantially all of C.I. brighteners 260 are to be in alpha-crystal form.
Brightener typically be in micronized particulate form, with from 3 to 30 microns, from 3 microns to 20 microns or from 3 to 10 microns of weighted average primary particle size.
Said composition can include the C.I. brighteners 260 in β-crystal form, and (i) is in alpha-crystal shape The weight ratio of the C.I. brighteners 260 of formula and (ii) C.I. brighteners 260 for being in β-crystal form can be to Few 0.1 or at least 0.6.BE 680847 is related to the method for the C.I. brighteners 260 in alpha-crystal form to be made.
The commercial optical brightener that can be used in the present invention can be divided into multiple subgroups, and these subgroups include but are not It is necessarily limited to：Talan, pyrazoline, cumarin, carboxylic acid, methine cyanines, dibenzothiophenes -5,5- dioxide, azole, 5 yuan and the derivative of 6 membered ring heterocyclics and other mix agent.The example of such brightener is disclosed in the " production of brightener And application ", M. Zuo Helaodenike (Zahradnik), by New York John Wiley father and son company (John Wiley＆Sons, New York (1982)) are published.The specific non-limiting examples that can be used for the optical brightener of the present composition are in US Those identified in 4790856 and US 3646015.
Suitable brightener has following structure in addition：
Suitable brightener level is included from 0.01wt%, from 0.05wt%, from 0.1wt% or from 0.2wt%'s Reduced levels to 0.5wt% or 0.75wt% higher level.
On the one hand, brightener can be loaded on clay to form particle.The composition of silicate-present invention can be with Include silicate, such as sodium metasilicate or potassium silicate.Said composition can be included from 0wt% to less than 10wt% silicate, extremely 9wt% or to 8wt% or to 7wt% or to 6wt% or to 5wt% or to 4wt% or to 3wt% or even extremely 2wt%, and from higher than 0wt% or from 0.5wt% or from 1wt% silicate.Suitable silicate is sodium metasilicate.
DispersantThe composition of-the present invention can also include dispersant.Suitable water-soluble organic materials include homopolymerization Or the acid of combined polymerization or its salt, wherein polycarboxylic acids includes at least two carboxyls that are separated from each other by not more than two carbon atoms.
Enzyme stabilizers- the enzyme for being used to use in the composition can be by various technologies come stable.Enzyme as used herein can With the presence by calcium and/or the water-soluble source of magnesium ion come stable.The example of conventional stabilizer is such as polyalcohol (such as third Glycol or glycerine), sugar or sugar alcohol, peptide aldehyde, lactic acid, boric acid or boronic acid derivatives (such as aromatic boric acid ester) or phenylboric acid Derivative (such as 4- formylphenyl boronic acids), and said composition can be such as the institute in such as WO 92/19709 and WO 92/19708 Description is prepared.In the case of the waterborne compositions comprising protease, reversible protease inhibitors can be added, for example, are wrapped Include the boron compound of borate, 4- formyl phenylboronic acids, phenylboric acid and its derivative, or such as calcium formate, sodium formate and The compound of 1,2-PD, further to improve stability.Peptide aldehyde can have chemical formula B2-B1-B0- R, wherein：R be hydrogen, CH3、CX3、CHX2Or CH2X, wherein X are halogen atoms；B0It is that the phenylalanine for having OH substituents in contraposition and/or meta is residual Base；B1It is single amino acids residue；And B2It is made up of one or more amino acid residues, optionally including N- terminus protecting groups Group.It is preferred that peptide aldehyde include but is not limited to：Z-RAY-H、Ac-GAY-H、Z-GAY-H、Z-GAL-H、Z-GAF-H、Z-GAV-H、Z- RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H or Ac-WLVY-H, wherein Z are benzyloxies Base carbonyl and Ac is acetyl group.
Solvent- suitable solvent includes water and other solvents, such as lipophilic fluid.The example of suitable lipophilic fluid Including siloxanes, other silicone, hydrocarbon, glycol ether, glycerol derivatives (such as glycerin ether), fluoridized amine, it is fluoridized and Hydrofluoroether solvent, the organic solvent of low volatility nonfluorinated, diol solvent, other environmentally friendly solvents and its mixture.
Structural agent/thickener- structuring liquid can from internal structured, thus structure by primary sector (for example, table Face surfactant material) formed, and/or provided by using secondary component (for example, polymer, clay and/or silicate material) Three dimensional matrix structure and from external structurant.Said composition can include from 0.01wt% to 5wt% or from 0.1wt% to 2.0wt% structural agent.The structural agent is typically chosen from the following group, and the group is made up of the following：Diglyceride and glycerine Three esters, stearic acid diethylene glycol dilaurate, microcrystalline cellulose, the material based on cellulose, microfibrous cellulose, the alkalescence of hydrophobically modified Expandable emulsion (such as Polygel W30 (3VSigma)), biopolymer, xanthans, gellan gum and its mixture. Suitable structural agent includes the castor oil and its unethoxylated derivative of hydrogenation.Suitable structural agent is disclosed in US In 6855680.Such structural agent has screw thread spline structure system, and the system has a series of aspect ratios.Other are suitable Structural agent and it is described in for their method to be made in WO 10/034736.
Conditioning agentThe composition of-the present invention can include hard fat compound.Useful hard fat herein Compound has 25 DEG C or higher of fusing point, and is selected from the group, and the group is made up of the following：Fatty alcohol, aliphatic acid, fatty alcohol Derivative, derivative of fatty acid and its mixture.Such compound with low melting point is not intended to be incorporated herein part In.The non-limiting examples of high melting compound are found in international cosmetic ingredient dictionary (International Cosmetic Ingredient Dictionary), the 5th edition, 1993, and CTFA Cosmetic Ingredient Dictionaries (CTFA Cosmetic Ingredient Handbook), the second edition, in 1992.
In view of providing improved regulation benefit (wet and slippery sense, pliability such as during wet hair is applied to and to doing The moisturizing sense of hair), hard fat compound with from 0.1wt% to 40wt%, from 1wt% to 30wt%, from 1.5wt% to 16wt%, the level from 1.5wt% to 8wt% are included in the composition.
The composition of the present invention can include cationic polymer.The concentration of cationic polymer is typically in the composition Scope is from 0.05wt% to 3wt%, from 0.075wt% to 2.0wt% or from 0.1wt% to 1.0wt%.Used in expection Under the pH of said composition, suitable cationic polymer will have at least 0.5meq/gm, at least 0.9meq/gm, at least 1.2meq/gm, at least 1.5meq/gm or the cationic charge density less than 7meq/gm and less than 5meq/gm, the pH's Scope will be substantially from pH3 to pH9 or between pH4 and pH8.Here, " cationic charge density " of polymer refers to gather The ratio between molecular weight of positive charge number and polymer on compound.The mean molecule quantity of such suitable cationic polymer will be big It is between 10,000 and 10,000,000, between 50,000 and 5,000,000 or between 100,000 and 3,000,000 on body.
Suitable cationic polymer for using in the present compositions includes cation nitrogen moiety, for example Quaternary ammonium or cation protonated amino part.Any anionic counter ion can be associated with cationic polymer and used, only Want polymer to be held in solution in, in composition or in the condensed phase of composition, as long as and counter ion in physics and chemistry The upper main component with composition mutually perhaps will not inadequately damage composition properties, stability or U.S. otherwise Sense.The non-limiting examples of such counter ion include halide (for example, chloride, fluoride, bromide, iodide), sulfuric acid Salt and Methylsulfate.
The non-limiting examples of such polymer are described in CTFA cosmetic ingredient dictionaries, the third edition, Estlin (Estrin), Crosley (Crosley) and Haynes (Haynes) write (cosmetics, toiletry and perfume joint public affairs Take charge of (The Cosmetic, Toiletry, and Fragrance Ass °C iation, Inc.), Washington (1982)).
Other suitable cationic polymers for using in the composition include polysaccharide polymer, cation Guar Bean gum derivative, the cellulose ether of quaternary nitrogen containing, synthetic polymer, the copolymer of etherified cellulose, guar gum and starch.When When use, cationic polymer in this is dissolvable in water in composition or is dissolvable in water the complex coacervation phase in composition In, the condensed phase is cationic polymer and anion by mentioned earlier, both sexes and/or zwitterionic surface-active agent component Formed.The complex coacervation thing of cationic polymer can also be electrically charged with other in composition material formed.Suitable sun from Sub- polymer is described in US 3962418；US 3958581；In US 2007/0207109.
The composition of the present invention can include the non-ionic polymers as conditioning agent.There is the molecule more than 1000 herein The polyalkylene glycols (polyalkylene glycol) of amount are useful.Those with below general formula are useful：
Wherein R95It is selected from the group, the group is made up of the following：H, methyl and its mixture.Conditioning agent, and particularly Silicone, can be included in the composition.Typically comprise to form emulsifying liquid for the conditioning agent in the composition of the present invention Water-insoluble, water dispersible, the non-volatile liquid of particle.Suitable conditioning agent for using in the composition is logical Often it is characterized as those conditioning agents of following item：Silicone (for example, silicone oil, cation silicone, silicone adhesive, high refractiveness silicone, with And silicone resin), organic regulation oily (for example, hydrocarbon ils, polyolefin and fatty ester) or its combination, or exist in another manner Those conditioning agents of liquid dispersion particle are formed in this aqueous tenside matrix.Such conditioning agent should in physics and It is chemically compatible with the key component of composition, and it is stable inadequately to damage composition otherwise Property, aesthetic feeling or performance.
The concentration of conditioning agent should be enough to provide desired regulation benefit in the composition.This concentration can be with tune Save agent, desired regulation performance, the mean size of conditioning agent particle, the type of other components and concentration and other it is similar because Element and change.
The concentration range of silicone conditioning agent is typically from 0.01wt% to 10wt%.Suitable silicone conditioning agent and right The U.S. Reissue patent No. 34,584 is described in the non-limiting examples of the optional suspending agent of silicone；US 5104646；US 5106609；US 4152416；US 2826551；US 3964500；US 4364837；US 6607717；US 6482969；US 5807956；US 5981681；US 6207782；US 7465439；US 7041767；US 7217777；US 2007/ 0286837A1；US 2005/0048549A1；US 2007/0041929A1；GB 849433；, will be all in DE 10036533 Document is incorporated herein by reference；The chemistry and technology (Chemistry and Technology of Silicones) of silicone, New York：Academic press (1968)；General Electric silicone rubber product data list SE 30, SE 33, SE 54 and SE 76；Silicon Assimilation compound (Silicon Compounds), Petrarch system house (Petrarch Systems, Inc.) (1984)；With And polymer science and engineering encyclopedia (Encyclopedia of Polymer Science and Engineering), volume 15, second edition, the 204-308 pages, John Wiley father and son company (John Wiley＆Sons, Inc.) (1989) in.
At least one organic regulation oil that the composition of the present invention can also include from 0.05wt% to 3wt% is used as regulation Agent, is combined individually or with other conditioning agents such as silicone (described herein).Suitable regulation oil include hydrocarbon ils, polyolefin, with And fatty ester.It is in US 5674478 and US 5750122 or in US 4529586 to be suitable also in composition in this； US 4507280；US 4663158；US 4197865；US 4217914；US 4381919；And described in US 4422853 Conditioning agent.
Hygiene and foul smellThe composition of-the present invention can also include ricinoleic acid zinc, thymol, quaternary ammonium salt (for example), polyethyleneimine is (such as from BASF AG (BASF)) and its zinc complexes, silver and silver Compound (is especially designed to slowly discharge Ag+Or those of nanometer silver dispersions) in one or more.
Probiotic- these compositions can include probiotic, as being described in WO 09/043709.
Foam improverIf-high foaming is desirable to, foam improver (such as C10-C16Alkanolamide or C10-C14Alkylsurfuric acid Ester) can be typically with 1wt% to 10wt% level incorporation composition.C10-C14Monoethanol and diglycollic amide are elaborated The typical classification of such foam improver.Such foam improver is with high sudsing adjunct surfactants (for example, above-mentioned oxidation Amine, glycine betaine and sulfobetaines (sultaine)) it is used together and is also advantageous.If desired, water soluble magnesium And/or calcium salt (such as MgCl2、MgSO4、CaCl2、CaSO4Deng) typically can be added with 0.1wt% to 2wt% level, To provide other foam and to strengthen grease removal capacity.
Foam in hibitors- can be mixed in composition of the invention for reducing or suppressing the compound of formation of foam.Bubble Foam suppresses in such as so-called " the high concentration cleaning procedure " described in US 4489455 and US 4489574 and in front bearing formula (front-loading-style) it is probably especially important in rinsing maching.Diversified material may be used as foam suppression Agent, and foam in hibitors is well known for a person skilled in the art.See, e.g. Ke Keaosimo chemical industry hundred Section's pandect (Kirk Othmer Encyclopedia of Chemical Technology), the third edition, volume 7,430- Page 447 (John Wiley father and son company, 1979).The example of foam in hibitors includes mono carboxylic aliphatic acid and therein solvable Salt, high-molecular-weight hydrocarbons such as paraffin, fatty acid ester (for example, fatty acid triglycercide), the fatty acid ester of monovalent alcohol, aliphatic C18-C40Ketone (such as stearone), the preferably amino triazine of N- alkylations, the wax hydrocarbon with the fusing point under about 100 DEG C, silicone Foam in hibitors, and secondary alcohol.Foam in hibitors is described in US 2954347；US 4265779；US 4265779；US 3455839；US 3933672；US 4652392；US 4978471；US 4983316；US 5288431；US 4639489；US 4749740；US 4798679；US 4075118；EP 89307851.9；EP 150872；And in DOS 2,124,526.
For there is any detergent composition being ready to use in automatic washing machine, foam should not form their spillings and wash Wash the degree of machine.When deployed, foam in hibitors is preferably what is existed with " foam amount of suppression "." foam amount of suppression " means combination The makers-up of thing can select the amount of this foam controller, and this amount will fully control foam to cause to be used for automatic laundry Low foaming laundry detergent compositions in machine.
Composition in this will generally include foam in hibitors from 0 to 10wt%.When as foam in hibitors, single carboxylic Base aliphatic acid and salt therein typically will exist with up to 5wt% amount.Preferably, using from 0.5wt% to 3wt% Aliphatic mono-carboxylic acids' ester foam in hibitors.Silicone foam inhibitor is typically used with up to 2.0wt% amount, although can use Higher amount.Single stearoyl phosphate foam in hibitors is generally used with the amount of the scope from 0.1wt% to 2wt%.Typically Hydrocarbon foam in hibitors is used with the amount of the scope from 0.01wt% to 5.0wt%, although higher level can be used.Typically Alcohol foam in hibitors is used with 0.2wt% to 3wt%.
Composition in this can have cleaning action in the range of wide pH.In certain embodiments, these compositions With the cleaning action from pH4 to pH11.5.In other embodiments, these compositions from pH6 to pH11, from pH7 to pH11, It is from pH8 to pH11, from pH9 to pH11 or active from pH10 to pH11.5.
Composition in this can have cleaning action in the temperature (such as 90 DEG C from 10 DEG C or more as little as) of wide scope. Preferably, the temperature will be less than 50 DEG C or 40 DEG C or even 30 DEG C.In certain embodiments, for these compositions Optimum temperature range is from 10 DEG C to 20 DEG C, from 15 DEG C to 25 DEG C, from 15 DEG C to 30 DEG C, from 20 DEG C to 30 DEG C, from 25 DEG C to 35 DEG C, from 30 DEG C to 40 DEG C, from 35 DEG C to 45 DEG C or from 40 DEG C to 50 DEG C.
The form of composition
Composition described here be advantageously utilised in for example laundry applications, hard-surface cleaning, dishwashing detergent application, together with In cosmetic applications (such as artificial tooth, tooth, hair and skin).The present invention composition be specifically solid or cleaning liquid and/or Treatment compositions.On the one hand, the present invention relates to a kind of composition, the form of wherein said composition is selected from the group, the group by with Lower every composition：The liquid of rule, compression or concentration；Gel；Cream；Soap bar；Rule or compression powder；It is granular solid Body；Uniform or multilayer tablet with two or more layers (identical or different phase)；Bag with one or more rooms；It is single Or multiple room unit dosage forms；Or its any combinations.
The form of said composition can be by the component in multiple rooms (such as water soluble bag) or in the different layers of tablet Physically it is separated from each other.It is possible thereby to the negative storage interaction between avoiding component.In wash solution, each room Different solubility curves can also cause the delayed dissolved of the component of selection.
Bag can be configured as single or multiple rooms.It, which can have, is adapted to any form, shape that appearance holds said composition And material, such as before being contacted with water, do not allow said composition to be discharged from bag.Bag by encapsulation inner volume water solubility Film is made.The inner volume can be divided into the room with bag.It is preferred that film be the polymeric material to form film or piece, preferably gather Compound.It is preferred that polymer, copolymer or derivatives thereof be selected from polyacrylate and water-soluble acrylic ester copolymer, methyl Cellulose, carboxymethyl cellulose, dextrin sodium, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, Polymethacrylates, most preferably polyvinyl alcohol copolymer and hydroxypropyl methyl cellulose (HPMC).Preferably, it polymerize Level of the thing in film such as PVA is at least about 60%.It is preferred that mean molecule quantity will be typically about 20,000 to about 150,000.Film can also be blend composition, and the blend composition is blended including degradable and water soluble and water soluble polymer Thing, such as PLA and polyvinyl alcohol (it is known under trade reference M8630, such as by No. MonoSol of Indiana, USA Co., Ltd sells) plus plasticizer, as glycerine, ethylene glycol, propane diols, sorbierite and its mixture.These bags can be wrapped Include solid laundry Cleasing compositions or constituent part and/or liquid cleansing composition or the constituent part separated by water-solubility membrane. Room for liquid component in composition can be different from the room including solid (US 2009/0011970A1).
Lipase enzyme granule
Lipase Variant of the invention included in water-solubility membrane can exist as lipase enzyme granule.These lipase Particle can even include one or more other enzymes, as described below.
Lipase enzyme granule is any form of the lipase Variant in solid particulate.Lipase enzyme granule can be fat Fat enzyme crystal, lipase precipitation, spraying or lyophilized lipase or any type of granular lipase, in being powder or liquid Suspension.Typically, the granularity for being measured as the lipase enzyme granule of equivalent spherical diameter (particle mean size based on volume) is less than 2mm, excellent Choosing is less than 1mm, less than 0.5mm, less than 0.25mm or less than 0.1mm；And higher than 0.05 μm, preferably above 0.1 μm, be higher than 0.5 μm, higher than 1 μm, higher than 5 μm or higher than 10 μm.In a preferred embodiment, the granularity of lipase enzyme granule is from 0.5 μm To 100 μm.
Lipase enzyme granule includes at least 1%w/w Lipase proteins, preferably at least 5%w/w Lipase proteins, at least 10% W/w Lipase proteins, at least 20%w/w Lipase proteins, at least 30%w/w Lipase proteins, at least 40%w/w lipase egg In vain, at least 50%w/w Lipase proteins, at least 60%w/w Lipase proteins, at least 70%w/w Lipase proteins, at least 80% W/w Lipase proteins or at least 90%w/w Lipase proteins.
In a preferred embodiment, lipase enzyme granule is fatty enzyme crystal, or Lipase protein is to be in crystal form.
Enzyme crystallization can be carried out by various ways as known in the art (for example, being such as described in WO 91/09943 or WO In 94/22903).
Lipase can be formulated in lipase enzyme granule as known in the art, for solid agent prepared from enzyme, for example with In the preparation for the rate of release for reducing dust, improving stability and/or changing enzyme.Lipase enzyme granule can also be formulated in base In matter or it is coated with reagent, it is molten in the PVOH/ coating solutions for preparing water-solubility membrane that these matrix or reagent suppress enzyme granulate Solution.
Lipase molecules on the surface of lipase enzyme granule can also be crosslinking, as CLEC (crosslinked enzyme crystal) or CLEA (cross-linked enzyme aggregate).
Water-solubility membrane
Water-solubility membrane, for optional member therein and prepare their method and be well known in the art. In one class embodiment, the water-solubility membrane includes PVOH.PVOH is a kind of generally by alcoholysis (commonly known as hydrolysis or saponification) Polyvinyl acetate and the synthetic resin prepared.The PVOH of complete hydrolysis (wherein nearly all acetate has been converted into alcohol radical) It is a kind of highly crystalline polymer of the strong hydrogen bonding bonding of dissolving only in hot water (greater than about 140 °F (60 DEG C)).If poly- Allow the acetate of remaining number enough after vinyl acetate ester hydrolysis, then the PVOH polymer is referred to as partial hydrolysis, Its hydrogen bonding is weaker and crystallinity is lower and is solvable in cold water (being below about 50 °F (10 DEG C)).In the middle of a kind of Cold/hot water soluble film can include the PVOH (for example, degree of hydrolysis with about 94% to about 98%) of such as center section hydrolysis, And readily soluble only in warm water (for example, quickly being dissolved at a temperature of about 40 DEG C and the above).The PVOH fully and partially hydrolyzed Type is all commonly known as PVOH homopolymers, although the type of partial hydrolysis is technically a kind of vinyl alcohol-vinyl acetate Copolymer.
The degree of hydrolysis for the PVOH being contained in the water-solubility membrane of present disclosure can be about 75% to about 99%.When degree of hydrolysis drop When low, the film being formed from a resin dissolves faster at the temperature by the mechanical strength with reduction but below about 20 DEG C.When During degree of hydrolysis increase, the film being formed from a resin will tend to that mechanical strength is higher and hot formability will tend to reduction.Can be with The water solubility of the resin, which is temperature dependency, to be chosen so as to PVOH degree of hydrolysis, and therefore by resin, compatible The solubility for the film that property reagent and other composition are made also is affected.In a class embodiment, the film is cold water solubles 's.A kind of film of cold water solubles (in water-soluble at a temperature of less than 10 DEG C) can include degree of hydrolysis about 75% to about PVOH in the range of 90% or in the range of about 80% to about 90% or in the range of about 85% to about 90%.In another kind of reality Apply in example, the film is hot water soluble.A kind of film of hot water soluble (in water-soluble at a temperature of at least about 60 DEG C) can be with Including the PVOH that degree of hydrolysis is at least about 98%.
In addition to PVOH or in PVOH alternative solution, other used film-forming resins may include but be not limited to change Polyvinyl alcohol, polyacrylate, water-soluble acrylic resin copolymer, polyacrylate, polyacrylamide, the polyvinyl pyrrole of property Alkanone, Propiram, water-soluble natural polymer include but is not limited to guar gum, xanthans, carrageenan and starch, water-soluble Property polymer derivant include but is not limited to the starch of ethoxylation and the starch of hydroxypropylation, poly- (acrylamido -2- first Base propane sulfonic acid sodium), poly mono-methyl, its copolymer and the combination of any of the above described.In a class embodiment, this into Film resin is a kind of ternary polymerization being made up of vinyl alcohol, vinyl acetate and acrylamido -2- methyl propane sulfonic acid sodium Thing.Unexpectedly, the ternary polymerization based on vinyl alcohol, vinyl acetate and acrylamido -2- methyl propane sulfonic acid sodium The enzyme that the water-solubility membrane of thing has shown high percentage is reclaimed.
Water-soluble resin can be included by any suitable amount (such as the amount in the range of about 35wt% to about 90wt%) In the water-solubility membrane.The amount of the water-soluble resin is excellent compared with the combined amount of all enzymes, enzyme stabilizers and auxiliary additive Weight is selected than that can be any suitable ratio, such as the ratio in the range of about 0.5 to about 5 or about 1 to about 3 or about 1 to about 2 Rate.
For be in the film of this description the water-soluble resin (including, but are not limited to PVOH resins) that uses can by for Any viscosity being adapted to for desired membrane property is characterized, optionally in about 5.0 to about 30.0cP or about 10.0cP to about Viscosity in the range of 25cP.The viscosity of PVOH resins is by using Bu Shi (Brookfield) LV type viscosity with UL adapters Meter is determined measuring freshly prepd solution, such as in British Standard EN ISO15023-2:Institute in 2006 annex E Bu Shi methods of testing Description.The viscosity that 4% polyvinyl alcohol water solution is illustrated at 20 DEG C is international practice.Glued herein with the cP all PVOH specified Spend and should be understood to refer to the viscosity of 4% polyvinyl alcohol water solution at 20 DEG C, unless otherwise indicated.
It is rather largely known in the art that the viscosity of PVOH resins and the weight average molecular weight of same PVOH resins Correlation, and the viscosity is often used asAgency.Therefore, the weight average molecular weight of the water-soluble resin optionally may be used With in the range of about 35,000 to about 190,000 or about 80,000 to about 160,000.The molecular weight of the resin only needs to foot To cause it to be moulded to form plastic sheeting by appropriate technology.
Other optional additives are included according to the amount that the water-solubility membrane of present disclosure can for example be suitable for its expected purpose Composition, includes but is not limited to, plasticizer, surfactant, defoamer, film forming agent, anti-blocking agent, inner pattern releasing agent applicable, anti-yellowing agent And other functional components.
Water is considered as a kind of very effective plasticizer for PVOH and other polymers, however, the volatilization of water Property causes its compromised utility, because polymer film needs to have a variety of environmental conditions (including low and high relative humidity) At least some tolerances (robustness).The volatility of glycerine is more much smaller than water, and is asserted well for PVOH With a kind of effective plasticizer for other polymers.If the level used in film preparation is too high, glycerine or other This liquid plasticizer their own can cause surface " perspiring (sweating) " and greasy.This can cause following in film Problem, for example the hand to consumer is with unacceptable sense of touch, without (for example, surface dusting) in some way Mitigate and perspire, or even film can be blocked on roller or in thin slice in heaps.This can be characterized as plasticizing.If however, be added to Very little, the film may lack enough ductility and flexibility to many final uses to the plasticizer of film, for example, be converted to final Usage type, such as bag.
Plasticizer for being used in the water-solubility membrane of present disclosure includes but is not limited to sorbierite, glycerine, diglycerol, third Glycol, ethylene glycol, diethylene glycol (DEG), triethylene glycol, tetraethylene glycol, polyethylene glycol (up to MW 400), 2- methyl-1,3-propanediols, lactic acid, Monoacetin, glyceryl triacetate, triethyl citrate, 1,3 butylene glycol, trimethylolpropane (TMP), polyether triol and its group Close.As described above, polyalcohol generally can be used as plasticizer.Plasticizer use is fewer, and film may become more crisp, and plasticizer is used More, film may lose tensile strength.Plasticizer can by for example in about 25phr to about 50phr, or from about 30phr to about 45phr, or from about 32phr to about 42phr in the range of amount be included in the water-solubility membrane.
Surfactant for being used in water-solubility membrane is well known in the art.Optionally, including surface Activating agent with casting assisted resin solution it is scattered.Suitable surfactant for the water-solubility membrane of present disclosure includes But it is not limited to the Lactated fatty acid ester of dialkyl sulfosuccinates, glycerine and propane diols, aliphatic acid lactoyl ester, alkylsurfuric acid Sodium, polysorbate 20, polysorbate 60, polysorbate 65, polyoxyethylene sorbitan monoleate, alkyl polyoxyethylene ether, lecithin, glycerine and third Acetylated fatty acid esters, NaLS, fatty acid acetylation ester, myristyl dimethyl amine oxide, the trimethyl of glycol Tallow alkyl ammonium chloride, quaternary ammonium compound, its salt and the combination of any of the above described.Therefore, surfactant can be by for example Less than about 2phr, for example, less than about 1phr or the amount less than about 0.5phr are included in the water-solubility membrane.
Consider that a type of secondary component used is defoamer.Defoamer can aid in coalesced foam bubble.With Include but is not limited to hydrophobic silica, such as fine granularity in the suitable defoamer in the water-solubility membrane according to present disclosure Silica or fumed silica, including FoamDefoamer (is available from liking black Ruide high performance material company (Emerald Performance Materials)), including Foam327、FoamUVD、Foam 163、Foam269、Foam338、Foam290、Foam332、Foam349、 Foam550 and Foam339, they are proprietary non-mineral oil defoamers., can be by embodiment 0.5phr or less amount use defoamer, such as 0.05phr, 0.04phr, 0.03phr, 0.02phr or 0.01phr.It is preferred that Ground, in order to avoid stress whitening, will avoid the silica of significant quantity.
Include casting, blowing, extrusion or blowing extrusion, such as this area for preparing the method for water-soluble article (including film) In it is known.The embodiment that one class considers forms sign by water-solubility membrane described here by casting, such as by will herein The composition of description is mixed with water to produce aqueous mixture (such as the solution with optionally scattered solid), should to surface applied Mixture, and dry removing water to produce film.Similarly, can be by drying the mixture, while being limited as desired Shape to form other compositions.
In the embodiment that a class considers, the water-solubility membrane is formed by water soluble mixt of casting, wherein the water solubility Mixture is prepared according to following steps：
(a) additive (a kind of mixture of water-soluble resin, water and any optional exclusion plasticizer is provided；
(b) boiling mixture 30 minutes；
(c) in an oven at least 40 DEG C；, will at a temperature of e.g., from about 65 DEG C optionally in the range of 40 DEG C to 70 DEG C Mixture deaerates；
(d) one or more enzymes, plasticizer and other water are added into mixture under 65 DEG C or lower temperature； And
(e) mixture is stirred in the case of without vortex, until the color and denseness of mixture seem substantially uniform； Optionally continue a period of time in the range of 30 minutes to 90 minutes, optionally at least 1 hour；And
(f) the rapid casting mixture (for example, in 4 hours or 2 hours or 1 hour) after the period of stirring.
If enzyme prematurely is added into mixture (for example, together with auxiliary additive or resin), enzymatic activity may drop It is low.It is not intended to bound by any particular theory, it is believed that the mixture boiled with enzyme is caused into enzyme denaturation and in the solution The period of storage extension again results in enzymatic activity reduction.
In a class embodiment, the water solubility according to present disclosure is caused by rapid desciccator diaphragm under moderate to temperate condition Film keeps high enzymatic activity.As used herein, rapid dry refers to be less than 24 hours, optionally less than 12 hours, optionally Less than 8 hours, optionally less than 2 hours, optionally less than 1 hour, optionally less than 45 minutes, optionally less than 30 minutes, It is optionally less than 20 minutes, optionally less than 10 minutes, such as dry in the range of about 6 minutes to about 10 minutes or 8 minutes The dry time.As used herein, moderate to temperate condition refers to the drying temperature less than 170 °F (77 DEG C), optionally about In the range of 150 °F to about 170 °F (about 66 DEG C to about 77 DEG C), for example, 165 °F (74 DEG C).As drying temperature increases, enzyme inclines Xiang Yuyue is denatured soon, and as drying temperature is reduced, thus drying time increase makes enzyme within the period of extension exposed to molten In liquid.
The film has for producing a kind of bag with comprising a kind of composition, for example, clothes washing or dish washing compositions, by This forms a kind of bag.Film described here can also be used to prepare a kind of bag with two or more compartments, and it is by identical The film of other polymeric materials of film or combination is made.Other film can for example be extruded by casting, blowing, extrusion or blowing Identical or different polymeric material and obtain, as known in the art.In the embodiment of a type, it is suitable for use as The polymer of other film, copolymer or derivatives thereof are selected from polyvinyl alcohol, polyvinylpyrrolidone, polyalkylene oxide, polypropylene Acid, cellulose, cellulose ether, cellulose esters, cellulose amides, polyvinyl acetate, polycarboxylic acids and salt, polyaminoacid or peptide, Polyamide, polyacrylamide, the copolymer of maleic acid/acrylic acid, polysaccharide (including starch and gelatin), natural gum (such as xanthan Glue and carrageenan).For example, polymer can selected from polyacrylate and water-soluble acrylic ester copolymer, methylcellulose, Sodium carboxymethylcellulose, dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, poly- methyl Acrylate and combinations thereof, or selected from polyvinyl alcohol, polyvinyl alcohol copolymer and hydroxypropyl methyl cellulose (HPMC) and its group Close.
The bag of present disclosure and/or include at least one sealed compartment.Therefore, these bags can include single compartment or Multiple compartments.These bags can have the region containing enzyme and without enzyme.In the embodiment including multiple compartments, each compartment can To include identical and/or different compositions.And then, these compositions can take any suitable form, including but not It is limited to, liquid, solid and combinations thereof (for example, the solid floated on a liquid).In certain embodiments, these bags include the First, second and the 3rd compartment, each of which compartment includes a kind of first, second, and third different composition respectively.One In a little embodiments, such as it is described in EP 2258820, these compositions can be visually different.
These compartments of multiple compartment pouch and/or bag can have one or more same or different sizes and/or body Product.These compartments of multiple compartment pouch of the present invention can be separated or combined in any suitable manner.In certain embodiments, This second and/or the 3rd and/or subsequent compartment be superimposed upon in the first compartment.In one aspect, the 3rd compartment can be folded It is added in the second compartment, the second compartment and then is superimposed upon with a kind of sandwich configuration in the first compartment.Alternately, this Two and the 3rd compartment can be superimposed upon in the first compartment.However, it is also possible to similarly it is contemplated that this first, second and appoint Selection of land the 3rd and subsequent compartment can be attached to each other by relation side by side.These compartments can be packaged into a string, each every Room is respectively separable by perforation line.Therefore, each compartment can be independent from the remainder of the string by terminal user Ground is torn off.
In certain embodiments, multiple compartment pouch and/or three compartments are included, it is by a big first compartment and two Less compartment composition.Less second and the 3rd compartment be superimposed upon in big first compartment.Size to these compartments and several What structure is selected, make it that this arrangement is achievable.The geometry of these compartments can be identical or different. In some embodiments, compared with the first compartment, this second each has different geometry knot with optionally the 3rd compartment Structure and shape.In these embodiments, this second and the optionally the 3rd compartment with one kind design be arranged in the first compartment. The design can be ornamental, teaching or illustrative, such as with one concept of explanation or guidance, and/or for referring to Show the source of the product.In certain embodiments, the first compartment is maximum compartment, and it is with two big close around periphery The face of envelope, and the second compartment is smaller, it covers the table in a face for being less than about 75% or the first compartment less than about 50% Area.In the embodiment that there are the 3rd compartment, said structure can be identical, but this second and the 3rd compartment Covering is less than about the surface area in a face of 60% or the first compartment less than about 50% or less than about 45%.
The bag and/or bag of present disclosure can include one or more different films.For example, in the embodiment of single compartment In, the bag can be folded by one and is made to itself and in the sealed wall in edge, or alternately, by two The edge wall that is sealed is made.In the embodiment of multiple compartments, the bag can be made up of one or more films, to cause Any given bag compartment can include the wall being made by single film or with the different multiple films constituted.On the one hand, more every Room bag includes at least three walls：One outer upper wall；One outer lower wall；And a partition wall.The outer upper wall and the outer lower wall lead to It is often outside that is relative and forming this bag.The partition wall the inside of this bag and along seal line be fixed to these lead to Often relative outer wall.The inside of the multiple compartment pouch is separated at least one first compartment and a second compartment by the partition wall. In a class embodiment, the partition wall can be that thus unique film comprising enzyme minimizes exposure of the consumer to these enzymes.
Bag and bag can be made of any suitable apparatus and method.For example, single compartment pouch can be stood with well known in the art Formula filling, horizontal filling or rotary drum filling technique are made.Such technique can be continuous or interruption.The film can be soaked And/or heat to improve its ductility.This method can also relate to using vacuum the film being drawn to a suitable mould In.Once the film is on the horizontal component on the surface, the vacuum that the film is drawn in the mould can implement about 0.2 to about 5 seconds or about 0.3 to about 3 or about 0.5 to about 1.5 second.The vacuum may be such that it is provided for example at 10 millibars to 1000 Millibar in the range of or one in the range of 100 millibars to 600 millibars push.
These moulds (bag can be made wherein) can have any shape, length, width and depth, depending on these bags Required dimension.If desired, these moulds can also be different in size and shape each other.For example, final bag Volume can be about 5ml to about 300ml or about 10 to 150ml or about 20 to about 100ml, and the big I phase of these moulds It should be adjusted.
On the one hand, the first and second sealed compartments are included.Generally, the second compartment is in first sealed compartments A kind of overlaying relation, to cause second sealed compartments and first sealed compartments to share a partition wall of this bag of inside.
On the one hand, including the bags of the first and second compartments further comprises the 3rd sealed compartments.Generally, the 3rd sealing Compartment is in a kind of overlaying relation with first sealed compartments, to cause the 3rd sealed compartments and first sealed compartments to share One partition wall of this bag of inside.
In different aspect, the first chamber and the second chamber are selected from one of following combination：Liquid, liquid；Liquid, Powder；Powder, powder；And powder, liquid.
In different aspect, first, second, and third composition is selected from one of following combination：Solid, liquid, liquid；With And liquid, liquid, liquid.
On the one hand, the single compartment or multiple sealed compartments include a kind of composition.The plurality of compartment can be wrapped each Containing identical or different composition.Said composition is selected from a kind of liquid, solid or its combination.
In the method, heat can be applied to the film, commonly referred to as thermoforming.It can be applied with any suitable method Heat.For example, before the film is supplied on surface or once on the surface, can by make it be under heating element heater or Directly it is heated by hot-air.Alternately, for example, can be by heating surface or a kind of hot article being applied into this It is heated indirectly on film.Infrared light can be used to heat the film.The film can be heated at least 50 DEG C, for example, about 50 To about 150 DEG C, about 50 to about 120 DEG C, about 60 to about 130 DEG C, about 70 to about 120 DEG C or about 60 to about 90 DEG C of temperature.
Alternately, the film can be soaked by any suitable means, for example, before it is supplied on surface or Once on the surface, directly by the way that (a kind of wetting agent is included into solution, the increasing for the film composition of water, the film composition Modeling agent or any combinations of foregoing item) it is sprayed on the film, or indirectly by wetting surface or by the way that one kind moistening article is applied It is added on the film.
Once film is heated and/or soaks, it can be drawn in an appropriate mould, it is preferred to use true It is empty.For example, the film can by least about 1.5 draw ratio, and for example, optionally be up to 2 draw ratio thermoforming.It can lead to Cross the filling that the molded membrane is completed using any suitable means.In certain embodiments, most preferred method will depend on production Product form and required filling speed.In certain embodiments, the molded membrane is filled by online filling technique.Then pass through Any suitable method, is closed the opening being filled through encapsulating using the second film, to form bag.This, which can work as, is horizontal simultaneously And completed in continuously and smoothly moves.Closing can be accomplished by the following way：Continuously by the second film (preferably water-solubility membrane) These opening sides of wrapping are supplied to above, and are then preferably sealed the first and second films together, typically mould it Between region and therefore between bag.
Any suitable sealed bundle and/or the method for its individual compartment can be utilized.The non-limiting examples of this kind of means Including heat seal, solvent welding, solvent seal or fluid-tight and combinations thereof.Can be at a temperature of at least 200 °F (93 DEG C), such as In about 220 °F (about 105 DEG C) to about 290 °F (about 145 DEG C) or the model of about 230 °F (about 110 DEG C) to about 280 °F (about 140 DEG C) Enclose it is interior by the water solubility bag and/or its individual compartment heat seal.Typically, sealed area will only be formed with heat or solvent processing Domain.Typically, heat or solvent can be applied on closed material by any method, and typically, are only applied to shape Into on sealed region.If using solvent seal or fluid-tight or welding, it may be preferable that same to apply heat.It is preferred that fluid-tight Or solvent including being optionally applied on the region between mould or closed material, by example by solvent seal/welding method Such as sprayed or be printed onto on these regions, and then applied pressure on these regions, to form sealing.For example, can With using sealed roller as described above and with (optionally also providing heat).
It is then possible to which the bag formed is cut by cutter device.Any known method can be used to complete cutting.Can Preferably, can also be cut by continuation mode, and preferably with constant speed and preferably when in water When prosposition is put.The cutter device can be for example a kind of sharp objects or a kind of hot article or laser, thus, in the feelings of the latter Under condition, the hot article or laser " burning " wear the film/sealing area.
The different compartments of multiple compartment pouch can be together made by pattern side by side, wherein resulting disjunctor bag can be by cutting Cut and separated or can not be separated.Alternately, these compartments can be separately made.
In certain embodiments, bag can be made according to a kind of method comprised the following steps：
A) first compartment (as described above) is formed；
B) recess is formed in some or all of the enclosed compartment formed in step (a), this is superimposed upon to produce one The second moulded compartments on first compartment；
C) filled by tertiary membrane and close the second compartment；
D) first, second, and third film is sealed；And
E) these films are cut to produce multiple compartment pouch.
The recess formed in step (b) can be realized by the compartment applying vacuum prepared into step (a).
In certain embodiments, second and/or the 3rd compartment can be made in separate steps, and then with this One compartment is combined, and is such as described in EP 2088187 or WO 2009/152031.
In other embodiments, bag can be made according to a kind of method comprised the following steps：
A) optionally using heat and/or vacuum, the first film formation first compartment is used on the first forming machine；
B) first compartment is filled with first chamber；
C) on the second forming machine, optionally deform the second film using heat and vacuum, to be made second and optionally the Three moulded compartments；
D) fill this second and the optionally the 3rd compartment；
E) using tertiary membrane seal this second and the optionally the 3rd compartment；
F) by this sealed second and the optionally the 3rd compartment be put into the first compartment；
G) seal this first, second and the optionally the 3rd compartment；And
H) these films are cut to produce multiple compartment pouch.
The adaptability that above method can be carried out based on first and second forming machine is selected it.In some implementations In example, first forming machine is preferably a kind of Horizontal molding machine, and second forming machine is preferably a kind of rotary drum forming machine, preferably Ground is located on first forming machine.
It should be appreciated that by using appropriate feed station, it is possible to a variety of different or unique combination things of manufacture incorporation And/or the multiple compartment pouch of different or unique liquid, gel or paste composition.
The method for manufacturing composition
The composition of the present invention can be configured to any suitable form, and can be selected by the person of being formulated Where prepared by method, and the non-limiting examples of these methods are described in the example of applicant and US 4990280；US 20030087791A1；US 20030087790A1；US 20050003983A1；US 20040048764A1；US 4762636； US 6291412；US 20050227891A1；EP 1070115A2；US 5879584；US 5691297；US 5574005；US 5569645；US 5565422；US 5516448；US 5489392；In US 5486303, all documents are incorporated by reference Herein.The composition or composition prepared in accordance with the present invention of the present invention includes cleaning and/or treatment compositions, said composition bag Include but be not limited to use in and fabric, hard surface and the composition on any other surface are handled in fabric and household care field, The fabric and household care field include：Air care (including air freshener and smell delivery system), car care, tableware Washing, fabric-conditioning (including softening and/or pure and fresh), clothes washing, laundry and rinsing additive and/or nursing, hard surface Cleaning and/or processing (including floor and Closestool cleanser), particle or powder type is universal or " weight dirt " detergent, especially Cleaning detergent；Liquid, gel or the universal detergent of cream form, especially so-called heavy-filth liquid type；Liquid is fine Fabric detergent；Manual dish washing detergent or light dirty dish washing detergent, especially high bubbling type those；Machine dishwashing detergent Agent, including for the different tablets used for family and public organizations, particle, liquid and rinse aid type：Automobile or ground Blanket shampoo, bathroom detergent (including Closestool cleanser)；And cleaning adjuvant, for example bleach additive and " decontamination rod " or Pre-process type, load the composition (lamella for for example adding drier) of matrix.It is preferably used for cleaning and/or handles spinning The composition and method of fabric and/or hard surface (being most preferably textile).Composition is preferably in the pre- of washing process The composition in process step or (being most preferably for textile washing step) in main wash step and using.
As used herein, term " fabric " and/or hard surface cleaning and/or treatment compositions " it is cleaning and treatment group The subset of compound, unless otherwise noted, the subset includes particle or powder type is universal or " weight dirt " detergent, especially clearly Clean detergent；The universal detergent of liquid, gel or pasty state, especially so-called heavy-filth liquid type；Liquid high-count fabric Detergent；Manual dish washing detergent or light dirty dish washing detergent, especially high bubbling type those；Machine dish washing detergent, Including for the different tablets used for family and public organizations, particle, liquid and rinse aid type；Cleaning liquid and disappear Toxic agent, automobile or carpet shampoos, bathroom detergent (including Closestool cleanser)；Fabric regulating composition (including soften and/or clear Newly), liquid, solid and/or drier sheet form be may be at；Together with cleaning adjuvant, for example, bleach additive and " decontamination Rod " or pretreatment type, the composition (lamella for for example adding drier) for loading matrix.All applicable such combinations Thing can be in standard, concentration or even highly concentrated form, or even can be in some aspects to such composition In non-aqueous degree.
Application method
Present invention resides in fabric and/or household care field be used for clean any surface (including processing textile or Crust or other surfaces) method.Consider cleaning as mentioned can be small-scale (in such as family's housework) and On a large scale two kinds (such as in such as industry and professional environment).In one aspect of the invention, this method is included in washing process Pre-treatment step or main wash step (are most preferably in textile washing step using or are alternatively used in tableware Used in washing (both including manual and automatic/mechanical dishwashing detergents)) in contact pending processing surface the step of.At this The one side of invention, lipase Variant and other components are sequentially added in the method for cleaning and/or handling surface. Alternately, lipase Variant and other components are simultaneously added.
As used herein, washing includes but is not limited to clean and mechanical agitation.Washing can be carried out with foam compositions (as described in WO 08/101958), and/or it is used as scouring and churned mechanically by applying alternating pressure (pressure/vacuum) Addition method or alternative are carried out.Such surface or fabric are dried can be by adopting in family or industrial environment Any of universal means completes.The Cleasing compositions of the present invention are preferably suited for should in laundry and dishwashing detergent Used in.Therefore, the present invention includes being used for the side of cleaning objects (including but is not limited to fabric, tableware, cutter and kitchen tools) Method.This method includes making the step of object to be cleaned is contacted with the Cleasing compositions, and the Cleasing compositions include applicant Cleasing compositions, cleaning additive or its mixture at least one aspect.Fabric can include can be in Conventional consumer Or the most of any fabrics being washed under public organizations' use condition.The solution can have the pH from 8 to 10.5.Can be Composition is used with the concentration from 500ppm to 15.000ppm in solution.Water temperature range is typically from 5 DEG C to 90 DEG C.Water with The ratio between fabric is typically from 1:1 to 30:1.
On the one hand, the present invention relates to a kind of method of the variant of use parent lipase, the variant, which is included in, to be corresponded to SEQ ID NO:Substitution at the E1C and N233C of 2 mature polypeptide position, with lipase active, and it is fatty with parent The mature polypeptide of enzyme has at least 60% but the sequence identity less than 100%.
On the one hand, should the present invention relates to the purposes that the composition of the variant comprising parent lipase is used for cleaning objects Variant, which is included in, corresponds to SEQ ID NO:Substitution at the E1C and N233C of 2 mature polypeptide position, with lipase activity Property, and have at least 60% with the mature polypeptide of parent lipase but be less than 100% sequence identity.
On the one hand, the parent lipase is a kind of lipase, and the lipase is the polypeptide with following amino acid sequence, The amino acid sequence：(a) with the wild type fat from Humicola lanuginosa (Humicola lanuginosa) strain DSM 4109 Enzyme has at least 90% uniformity；(b) compared with the wild type lipase, it is included in E1's or Q249Interior three-dimensional Electroneutral or negatively charged amino acid are by the substitution of positively charged amino acid at the surface of structure；In C-terminal include (c) Peptide is added；And/or (d) meets following limitation：(i) negative electrical charge amino is included in the position E210 of the wild type lipase Acid；(ii) negatively charged amino acid is included in the region of the position 90-101 corresponding to the wild type lipase；And (iii) include at the position of the N94 corresponding to the wild type lipase neutral or negative electrical charge amino acid and/or corresponding to There is negative net charge or neutral net charge in the position 90-101 of wild type lipase region.On the one hand, the parent Lipase is the lipase with lipase active, with SEQ ID NO:2 sequence with least 60% but less than 100% is consistent Property, and corresponding to SEQ ID NO:2 T231R+N233R and D96E, D111A, D254S, G163K, P256T, G91T, Include substitution at least one or more (for example, several) position in D27R and G38A.On the one hand, the parent lipase With SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino acid sequence.On the one hand, the parent lipase wraps The NO of ID containing SEQ:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide is made from it.
On the one hand, the present invention relates to a kind of method for producing said composition, this method includes addition parent lipase Variant and surfactant, the variant, which is included in, corresponds to SEQ ID NO:At the E1C and N233C of 2 mature polypeptide position Substitution, there is at least 60% but sequence less than 100% with lipase active, and with the mature polypeptide of parent lipase Uniformity.On the one hand, the present invention relates to the method for clean surface, this method includes making present on surface to be cleaned Lipid spot is contacted with the Cleasing compositions.On the one hand, the present invention relates to for hydrolyze be present in dirt on surface and/or The method of lipid in spot, this method includes making dirt and/or spot contact with Cleasing compositions.On the one hand, it is of the invention It is related to purposes of the said composition in carboxyester hydrolysis.On the one hand, the present invention relates to said composition ester hydrolysis, synthesis or Purposes in interesterification.On the one hand, the purposes for manufacturing stable formulations is used for the present invention relates to said composition.
On the one hand, the present invention relates to the purposes of the variant of parent lipase, the variant, which is included in, corresponds to SEQ ID NO:Substitution at the E1C and N233C of 2 mature polypeptide position, with lipase active, and with parent lipase into Ripe polypeptide has at least 60% but the sequence identity less than 100%.On the one hand, the parent lipase is by SEQ ID NO:2； SEQ NO NO:4；SEQ ID NO:6；Or there is any fragment of lipase active to constitute or comprising them for it.On the one hand, Ester hydrolysis, synthesis or interesterification are used for according to the variant of the present invention；Carboxylic acid ester hydrolysis；Hydrolyze lipid, cleans surface； And/or production detergent composition.
Example
Example 1：Determine
P-nitrophenyl (pNP) is determined：
The hydrolysing activity of lipase can be determined as the kinetic determination of substrate by using p-nitrophenyl acyl ester.
100mM stock solution of these the following substrates in DMSO can be diluted to measure buffer solution (50mM Tris； pH 7.7；1mM 25 final concentration in 0.4%TritonX-100)：P-nitrophenyl butyrate (C4), p-nitrophenyl capronate (C6), p-nitrophenyl decylate (C10), p-nitrophenyl laurate (C12) and p-nitrophenyl palmitate (C16) (institute It both is from Sigma-Aldrich Danish company (Sigma-Aldrich Danmark A/S), Kirkebjerg All é 84,2605 Cloth Longde ratioCatalog number (Cat.No.)：C4：N-9876, C6：N-0502, C10：N-0252, C12：N-2002, C16：N-2752).
Can be by 50mM Hepes (pH 8.0)；10ppm TritonX-100；+/-20mM CaCl2In the present invention Lipase, parent lipase and appropriate control (such as buffer solution (feminine gender), LipolaseTM＆LipexTM (positive)) in By 0.01mg/ml；5×10-3mg/ml；2.5×10-4mg/ml；96 hole energy are added to 1.25 × 10-4mg/ml final concentration Agree (NUNC) flat board (catalog number (Cat.No.)：260836, Kamstrupvej 90, DK-4000, Roskilde) in substrate solution in.Can With in Spectra max 190 (Molecular Devices limited company (Molecular Devices GmbH), Bismarck woods (Bismarckring) No. 39, this inner riverside is than Bai Lahe 88400, Germany) on, with 10 seconds intervals, monitoring was by right at 405nm The p-nitrophenol that nitrobenzophenone acyl is hydrolyzed and discharged, continues 5 minutes.Variant can be lived to the hydrolysis of one or more substrates Property is compared with parent lipase to the hydrolysing activity of one or more substrates.
Differential scanning calorimetry (DSC) is determined：
Using a VP- capillaries differential scanning calorimeter (micro hot company (Micr °C of al Inc.), Piscataway, New Jersey, the U.S.) heat endurance that (DSC) determines lipase Variant is determined by Differential Scanning Calorimetry.In 200K/hr perseverance Under fixed programmed heating rate, enzyme solutions (about 0.5mg/ml) in heating buffer solution (50mM Hepes, pH 8) it Afterwards, the top of peak (main endothermic peak) will be denatured in the thermal analysis curue (Cp is to T) of acquisition as thermal denaturation temperature Td (DEG C).
Under 10 DEG C of condition of storage, sample solution and reference solution (about 0.2mL) are loaded into calorimeter (reference Solution：Buffer solution without enzyme), and before the DSC scannings from 20 DEG C to 100 DEG C, hot 20 points of the pre-equilibration at 20 DEG C Clock.Denaturation temperature is determined with about +/- 1 DEG C of accuracy.
Also DSC is carried out by adding 0.05g/L Relase (Novozymes Company) and/or 1mM LAS.
Storage stability is determined：
Option A：Make the aspergillus oryzae strain of the generation lipase Variant at 37 DEG C in the 2xSC culture mediums with 2% maltose In without shaking grow 5 days.2xSC culture mediums are dissolved in the yeast nitrogen without amino acid of the 15g in 1L deionized waters (Difco 291920), 22.6g butanedioic acids (Merck ＆ Co., Inc. (Merck) 822260), 13.6g+ sodium hydroxides (Merck ＆ Co., Inc. 106498), 11.2g casamino acids (vitamin is determined, Difco 228830) and 0.2g L-Trps (Merck ＆ Co., Inc. 108374).
The 10uL nutrient solution is added into 90uL detergent compositions, is stirred 10 minutes, and is sealed in small plastics and is held In device.For stressed condition, at -20 DEG C, the C1 samples with detergent composition D0001 are stored in 0.02% chlorine Change calcium (stress not) detergent D001 in, and 48 DEG C (stress) under, be stored in 0.02% calcium chloride with In 1.35%Relase 16L EXI (Novozymes Company) detergent D001.For stressed condition, at -20 DEG C, will have Detergent composition D0001 C2 samples be stored in 0.02% calcium chloride (stress not) detergent D001 in, and 35 DEG C (stress) under, it is stored in 0.02% calcium chloride and 1.35%Savinase Ultra 16L (Novozymes Company) Detergent D001 in.For stressed condition, at -20 DEG C, the C3 samples with detergent composition D0002 samples are stored With 0.02% calcium chloride (stress not) detergent D002 in, and 55 DEG C (stress) under, being stored in has In 0.02% calcium chloride and 1.35%Relase 16L EXI (Novozymes Company) detergent D002.For stressed condition ,- At 20 DEG C, by the C4 samples with detergent composition D0002 samples be stored in 0.02% calcium chloride (stress not) wash Wash in agent D002, and 35 DEG C (stress) under, be stored in 0.02% calcium chloride and 1.35%Savinase16L In the detergent D002 of (Novozymes Company).Storage time is 19 hours.
After storage, it would be possible to condensed fluid by being collected by centrifugation.To 100uL stress or stress not sample in Add 230uL buffer solution (0.1M Tris-HCl；9mM CaCl2；0.0225%Brij-30；PH8.0+0.85%4-FBPA (31.5g/l)), this corresponds to 3.3 times of dilutions.After stirring in 10 minutes, 5uL sample aliquots are further delayed with identical Fliud flushing dilutes 60 times.Then by a this lipase dilution and four parts of 0.5mM pNP- palmitates, 1mM calcium chloride, 100mM Tris (pH8.0), 6.5mM dexycholates, 1.4g/L AOS mixing, and survey with continuing 30 minutes spectrophotometers Measure the release of pNP chromophores.This is used to determine activity by the initial linear slope of the reaction.
By residual activity (RA) be calculated as stress sample relative to stress not sample measuring speed ratio.Based on following formula meter Calculate half-life period (T1/2)：
Half-life period=stress the time*Ln (0,5)/ln (residual activity).
Intermediate value and the half-life period of residual activity are calculated based on two to four repetitions.
By by the half-life period of the lipase Variant divided by with sequence SEQ ID NO:The half-life period of 2 parent lipase To calculate the half-life period improvement factor (HIF) of specific mutation.
Option b (the purifying enzyme in the detergent with protease)：After active site titration, the lipase of purifying is become Body is diluted to the concentration specified with buffer solution (10mM butanedioic acid+2mM CaCl2+0.02%Brij 35 are adjusted to pH6.5).Add Plus 10uL 100ppm lipase solutions are into 90uL detergent compositions, stir 10 minutes, and seal.For stress bar Part, at -20 DEG C, by the C1 samples with detergent composition D0001 be stored in 0.02% calcium chloride (stress not) In detergent D001, and 48 DEG C (stress) under, be stored in 0.02% calcium chloride and 1.35%Relase 16L In EXI (Novozymes Company) detergent D001.For stressed condition, at -20 DEG C, there will be detergent composition D0001 C2 samples be stored in 0.02% calcium chloride (stress not) detergent D001 in, and 35 DEG C (stress) under, by it It is stored in the detergent D001 with 0.02% calcium chloride and 1.35%Savinase Ultra 16L (Novozymes Company).Pin To stressed condition, at -20 DEG C, the C3 samples with detergent composition D0002 samples are stored in 0.02% chlorination Calcium (stress not) detergent D002 in, and 55 DEG C (stress) under, be stored in 0.02% calcium chloride and In 1.35%Relase 16L EXI (Novozymes Company) detergent D002.For stressed condition, at -20 DEG C, will have The C4 samples of detergent composition D0002 samples be stored in 0.02% calcium chloride (stress not) detergent D002 in, and And 35 DEG C (stress) under, be stored in 0.02% calcium chloride and 1.35%Savinase 16L (Novozymes Company) In detergent D002.Storage time is 19 hours.
After storage, it would be possible to condensed fluid by being collected by centrifugation.To 100uL stress or stress not sample in Add 230uL buffer solution (0.1M Tris-HCl, 9mM CaCl2,0.0225%Brij-30, pH8.0+0.85%4-FBPA (31.5g/l)), this corresponds to 3.3 times of dilutions.After stirring in 10 minutes, 5uL sample aliquots are further delayed with identical Fliud flushing dilutes 60 times.Then by a this lipase dilution and four parts of 0.5mM pNP- palmitates, 1mM calcium chloride, 100mM Tris (pH8.0), 6.5mM dexycholates, 1.4g/L AOS mixing, and survey with continuing 30 minutes spectrophotometers Measure the release of pNP chromophores.This is used to determine activity by the initial linear slope of the reaction.
By residual activity (RA) be calculated as stress sample relative to stress not sample measuring speed ratio.Based on following formula meter Calculate half-life period (T1/2)：
Half-life period=stress the time*Ln (0,5)/ln (residual activity).
Based on four intermediate values for computing repeatedly residual activity and half-life period.Based on three intermediate values for computing repeatedly residual activity And half-life period.Calculating half-life period between a pair of different variants in single mutation or double mutation improves into the factor (HIF).With volume Outer single mutation or the half-life period of the variant of double mutation divided by the half-life period of variant without extra single mutation or double mutation It is half-life period improvement factor.
Detergent：Composition D001 is the commercially available AVA liquid from Reckitt Benckiser Co., Ltd (Reckitt Benckiser) Detergent.Composition D002 is the typical detergent being listed below.
With respect to scourability, RP (washing)
In order to assess the scourability in clothes washing, washing is carried out using automatic mechanical stress measurement (AMSA) real Test.AMSA flat boards have many seams and lid for being used to test solution, and lid is for the seamed opening strength extruding washing sample of institute (textile to be washed).During wash time, plate, test solution, textile and lid high vibration are so that test is molten Liquid contacts with textile and applies mechanical stress with rule, periodic oscillating manner.On further describing, referring to WO 02/ " ad hoc approach embodiment (Special method embodiments) " paragraph of 42740, especially the 23-24 pages.
Washing experiment is carried out using following experiment condition：
The paste turmeric dyeing of EMPA221 textiles is carried out according to WO 06/125437.With with paste turmeric dyeing EMPA221 identical modes produce the EMPA221 of paste roucou dyeing, except exchanging turmeric (A-320- with roucou WS is derived from Chr.Hansen Natural Colors A/S, Boege All é 10-12,2970 HessHolms, Denmark). EMPA221 is derived from EMPA companies, Lerchenfeldstrasse 5, CH-9014, holy gallon state (St.Gallen), Switzerland.
When being washed with detergent B, by adding CaCl2、MgCl2And NaHCO3(Ca2+:Mg2+:HCO3 -=4:1:7.5) The water hardness is adjusted to 5 ° of dH or 15 ° of dH, and when being washed with detergent X, the water hardness is adjusted to 12 ° of dH (Ca2+:Mg2+: HCO3 -=2:1:4.5).
Detergent B is the example of liquid detergent composition, and detergent X is the example of powder detergent composition.Wash After washing, textile is rinsed in running water and removed excessive water from textile using filter paper, and is stood afterwards Textile is dried into 15min at 100 DEG C.
The color change for the dirty textile that scourability is measured as being washed.When being washed with detergent B, by dirt with Roucou carries out paste mixing, and when being washed with detergent agent X, dirt and turmeric is carried out into paste and mixed.Arnotto It is red containing colouring agent norbixin (norbixin) and turmeric contains colouring agent curcumin.Norbixin and curcumin Worked by making pH carry out dependence color change as pH indicator.Lipase active causes free fatty from butterfat Acyl glyceride discharges and this causes pH to reduce and thus causes the color of norbixin or curcumin pH indicator to become Change.Therefore lipase scourability can be represented as when being illuminated with white light, from the dirty textile reflection-transmitting washed The discoloration of light.
Use the professional flatbed scanner (EPSON of the image of the textile for capturing washed pollution EXPRESSION 11000XL, Aunar Asia company (Atea A/S), the Barre Shandongs of Lautrupvang 6,2750 are general, Denmark) carry out Color measuring.In order to extract light intensity value from the image of scanning, 24 pixel values from image are converted into red, green With blue (RGB) value.
The color change for being attributed to lipase active is measured as the anti-of green light (G) relative to light intensity value (Int) The change penetrated-launched, the light intensity value by using rgb value it is added together as vector and and then take gained vector length come Calculate：
Relative to reference lipase, the relative scourability (RP (washing)) of lipase is calculated as：
RP (washing)=(G/Int (test lipase)-G/Int (no enzyme))/(G/Int (reference lipase)-G/Int (nothings Enzyme)).
If lipase performance is better than reference (RP (washing)>1), then it is assumed that lipase shows improved scourability. In the context of the present invention, reference enzyme is wild type lipase or parent lipase, i.e. pair without substitution E1C R233C According to lipase.
Nanometer differential scanning fluorescence (NanoDSF) is determined
Use nanometer differential scanning luminoscope (nanoDSF)；(nano temperature technical concern is limited by Prometheus NT.48 Company (NanoTemper Technologies GmbH), Munich, Germany) carry out heat endurance measurement.Use standard NanoDSF grades of capillaries (nano temperature technology companys (NanoTemper Technologies), catalog number (Cat.No.)：PR-C002).Will The protein example of purifying is loaded into capillary (each three parts of sample) by capillarity.By changing on instrument LED power optimize the emissive porwer at 330nm and 350nm, so as to obtain 3000 and 15000 fluorescence count between letter Number.From 20 degrees Celsius to 95 degrees Celsius, the temperature slope folded for being pyrolyzed is 3.3 degrees celsius/minutes.Carried using by manufacturer The software PR.Control v1.11.2 analyze datas of confession.Typically, just (or negative) peak in being analyzed using first derivative is maximum Value (or minimum value) represents thermal denaturation temperature Td (DEG C).
Directly using the sample in the liquid storage from purifying., will be in order to determine influence of the reducing agent to heat endurance 250mM TCEP (pH8.0) in 50mM HEPES are added in the sample that ultimate density is 0.5mM.Run parallel reduction and Sample under non reducing conditions.
Example 2：Stability
DSC according to example 1 determines to determine variant, reference lipase and prior art fat according to the present invention The stability of fat enzyme.The thermal denaturation temperature of every kind of lipase is shown in following table under the conditions of kind.
Table 2：Thermal denaturation temperature, Td
Under conditions of testing, lipase Variant of the invention shows stabilization more increased than prior art lipase Property.Also stability is have studied in the presence of 0.05g/L Relase (Novozymes Company) and/or 1mM LAS.Relase and/ Or in the presence of LAS, substitution E1C+N233C introducing result in stability increase.
Example 3：Stability
DSC according to example 1 determines to determine variant, reference lipase and prior art fat according to the present invention The stability of fat enzyme.The thermal denaturation temperature of every kind of lipase is shown in following table under the conditions of kind.
Table 3A：
Table 3B：
Table 3C：
Example 4：Storage stability
The variant replaced according to the option A test bag N233C containing E1C as described in example 1.With without E1C N233C The corresponding lipase of substitution is compared to show disubstituted influence.
Table 4：Improved stability
Example 5：Storage stability
The variant replaced according to the option b test bag N233C containing E1C as described in example 1.With without E1C N233C The corresponding lipase of substitution is compared to show disubstituted influence.
Table 5：Improved stability
Example 6：Scourability
Using detergent B with the technique study described in example 1 prior art lipase and the variant according to the present invention Relative scourability RP (washing), and the RP (washing) of itself and wild type lipase is compared.
Table 6：Compared to wild type and the improved scourability of prior art lipase.
Example 7：Scourability
Using liquid detergent, detergent B (Det B) or powder detergent, detergent X (Det X), retouched with example 1 The technique study stated is according to the relative scourability RP (washing) of the variant of the present invention, and by itself and corresponding parent lipase (the control lipase i.e. without substitution E1C+N233C) is compared.
Table 7：Compared to the improved scourability of parent lipase.
Example 8：Influence of the reducing agent to stability
NanoDSF according to example 1 determines to determine (not had according to variant of the invention, parent lipase Have E1C+N233C replace control lipase) and prior art lipase stability.The absence and presence of reducing agent (TCEP) in the case of, the thermal denaturation temperature of every kind of lipase is shown in following table.
Table 8：There is the stability improved with the absence of in reducing agent
It is described herein and claimed invention is not limited to the scopes of particular aspects disclosed here, because these Aspect is intended to the explanation as some aspects of the invention.It is expected that any equivalent aspect is all in the scope of the present invention.In fact, Except shown here and description in addition to those, different modifications of the invention are retouched from foregoing for those of ordinary skills State and will be clear.Such modification, which is also intended to, to be fallen within the scope of the appended claims.In case of conflict, with including The present disclosure of definition is defined.
Sequence table
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<213>Dredge the thermophilic hyphomycete of cotton like（Thermomyces lanuginosus）
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atg agg agc tcc ctt gtg ctg ttc ttt gtc tct gcg tgg acg gcc ttg 48
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10
gcc agt cct att cgt cga gag gtc tcg cag gat ctg ttt aac cag ttc 96
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 -1 1 5 10
aat ctc ttt gca cag tat tct gca gcc gca tac tgc gga aaa aac aat 144
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25
gat gcc cca gct ggt aca aac att acg tgc acg gga aat gcc tgc ccc 192
Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40
gag gta gag aag gcg gat gca acg ttt ctc tac tcg ttt gaa gac tct 240
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55
gga gtg ggc gat gtc acc ggc ttc ctt gct ctc gac aac acg aac aaa 288
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70
ttg atc gtc ctc tct ttc cgt ggc tct cgt tcc ata gag aac tgg atc 336
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile
75 80 85 90
ggg aat ctt aac ttc gac ttg aaa gaa ata aat gac att tgc tcc ggc 384
Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly
95 100 105
tgc agg gga cat gac ggc ttc act tcg tcc tgg agg tct gta gcc gat 432
Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120
acg tta agg cag aag gtg gag gat gct gtg agg gag cat ccc gac tat 480
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135
cgc gtg gtg ttt acc gga cat agc ttg ggt ggt gca ttg gca act gtt 528
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150
gcc gga gca gac ctg cgt gga aat ggg tat gat atc gac gtg ttt tca 576
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser
155 160 165 170
tat ggc gcc ccc cga gtc gga aac agg gct ttt gca gaa ttc ctg acc 624
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185
gta cag acc ggc gga aca ctc tac cgc att acc cac acc aat gat att 672
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200
gtc cct aga ctc ccg ccg cgc gaa ttc ggt tac agc cat tct agc cca 720
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215
gag tac tgg atc aaa tct gga acc ctt gtc ccc gtc acc cga aac gat 768
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
220 225 230
atc gtg aag ata gaa ggc atc gat gcc acc ggc ggc aat aac cag cct 816
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro
235 240 245 250
aac att ccg gat atc cct gcg cac cta tgg tac ttc ggg tta att ggg 864
Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly
255 260 265
aca tgt ctt tagtggccgg cgcggctggg tccgactcta gcgagctcga gatct 918
Thr Cys Leu
<210> 2
<211> 291
<212> PRT
<213>Dredge the thermophilic hyphomycete of cotton like
<400> 2
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 -1 1 5 10
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25
Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile
75 80 85 90
Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly
95 100 105
Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser
155 160 165 170
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
220 225 230
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro
235 240 245 250
Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly
255 260 265
Thr Cys Leu
<210> 3
<211> 1017
<212> DNA
<213>Artificial sequence
<220>
<223>Synthesize construct
<220>
<221> CDS
<222> (1)..(1017)
<220>
<221> sig
<222> (1)..(66)
<220>
<221>Signal peptide
<222> (1)..(66)
<220>
<221>Mature peptide
<222> (67)..()
<400> 3
atg agg agc tcc ctt gtg ctg ttc ttt gtc tct gcg tgg acg gcc ttg 48
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10
gcc agt cct ata cgt aga gag gtc tcg cag gat ctg ttt aac cag ttc 96
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 -1 1 5 10
aat ctc ttt gca cag tat tca gct gcc gca tac tgc gga aaa aac aat 144
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25
gat gcc cca gca ggt aca aac att acg tgc acg gga aat gcc tgc ccc 192
Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40
gag gta gag aag gcg gat gca acg ttt ctc tac tcg ttt gaa gac tct 240
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55
gga gtg ggc gat gtc acc ggc ttc ctt gct ctc gac aac acg aac aaa 288
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70
ttg atc gtc ctc tct ttc cgt ggc tct cgt tcc ata gag aac tgg atc 336
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile
75 80 85 90
gcg aat ctt aac ttc tgg ttg aaa aaa ata aat gac att tgc tcc ggc 384
Ala Asn Leu Asn Phe Trp Leu Lys Lys Ile Asn Asp Ile Cys Ser Gly
95 100 105
tgc agg gga cat gac ggc ttc act tcg tcc tgg agg tct gta gcc gat 432
Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120
acg tta agg cag aag gtg gag gat gct gtg agg gag cat ccc gac tat 480
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135
cgc gtg gtg ttt acc gga cat agc ttg ggt ggt gca ttg gca act gtt 528
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150
gcc gga gca gac ctg cgt gga aat ggg tat gat atc gac gtg ttt tca 576
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser
155 160 165 170
tat ggc gcc ccc cga gtc gga aac agg gct ttt gca gaa ttc ctg acc 624
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185
gta cag acc ggc gga aca ctc tac cgc att acc cac acc aat gat att 672
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200
gtc cct cga ctc ccg ccg cgc gaa ttc ggt tac agc cat tct agc cca 720
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215
gag tac tgg atc aaa tct gga acc ctt gtc ccc gtc acc cga aac gat 768
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
220 225 230
atc gtg aag ata gaa ggc atc gat gcc acc ggc ggc aat aac cag cct 816
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro
235 240 245 250
aac att ccg gat atc cct gcg cac ctg tgg tac ttc cag gcg act gac 864
Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gln Ala Thr Asp
255 260 265
gcc tgt aac gct ggt ggc ttc tct tgg cga cga tac aga agc gcc gag 912
Ala Cys Asn Ala Gly Gly Phe Ser Trp Arg Arg Tyr Arg Ser Ala Glu
270 275 280
agc gtc gac aag agg gcc acc atg act gat gcc gag ctt gag aag aag 960
Ser Val Asp Lys Arg Ala Thr Met Thr Asp Ala Glu Leu Glu Lys Lys
285 290 295
ctg aac tct tat gtc cag atg gat aag gag tat gtg aag aat aac cag 1008
Leu Asn Ser Tyr Val Gln Met Asp Lys Glu Tyr Val Lys Asn Asn Gln
300 305 310
gcc cgc tct 1017
Ala Arg Ser
315
<210> 4
<211> 339
<212> PRT
<213>Artificial sequence
<220>
<223>Synthesize construct
<400> 4
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 -1 1 5 10
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25
Asp Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro
30 35 40
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile
75 80 85 90
Ala Asn Leu Asn Phe Trp Leu Lys Lys Ile Asn Asp Ile Cys Ser Gly
95 100 105
Cys Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150
Ala Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser
155 160 165 170
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp
220 225 230
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro
235 240 245 250
Asn Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gln Ala Thr Asp
255 260 265
Ala Cys Asn Ala Gly Gly Phe Ser Trp Arg Arg Tyr Arg Ser Ala Glu
270 275 280
Ser Val Asp Lys Arg Ala Thr Met Thr Asp Ala Glu Leu Glu Lys Lys
285 290 295
Leu Asn Ser Tyr Val Gln Met Asp Lys Glu Tyr Val Lys Asn Asn Gln
300 305 310
Ala Arg Ser
315
<210> 5
<211> 878
<212> DNA
<213>Artificial sequence
<220>
<223>Synthesize construct
<220>
<221> CDS
<222> (1)..(873)
<220>
<221>Signal peptide
<222> (1)..(66)
<220>
<221>Mature peptide
<222> (67)..()
<400> 5
atg agg agc tcc ctt gtg ctg ttc ttt gtc tct gcg tgg acg gcc ttg 48
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10
gcc agt cct ata cgt aga gag gtc tcg cag gat ctg ttt aac cag ttc 96
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 -1 1 5 10
aat ctc ttt gca cag tat tct gca gcc gca tac tgc gga aaa aac aat 144
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25
agg gcc cca gct ggt aca aac att acg tgc acg gcc aat gcc tgc ccc 192
Arg Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Ala Asn Ala Cys Pro
30 35 40
gag gta gag aag gcg gat gca acg ttt ctc tac tcg ttt gaa gac tct 240
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55
gga gtg ggc gat gtc acc ggc ttc ctt gct ctc gac aac acg aac aaa 288
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70
ttg atc gtc ctc tct ttc cgt ggc tct cgt tcc ata gag aac tgg atc 336
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile
75 80 85 90
ggg aat ctt aac ttc gag ttg aaa gaa ata aat gac att tgc tcc ggc 384
Gly Asn Leu Asn Phe Glu Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly
95 100 105
tgc agg gga cat gcc ggc ttc act tcg tcc tgg agg tct gta gcc gat 432
Cys Arg Gly His Ala Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120
acg tta agg cag aag gtg gag gat gct gtg agg gag cat ccc gac tat 480
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135
cgc gtg gtg ttt acc gga cat agc ttg ggt ggt gca ttg gca act gtt 528
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150
gcc gga gca gac ctg cgt gga aat aag tat gat atc gac gtg ttt tca 576
Ala Gly Ala Asp Leu Arg Gly Asn Lys Tyr Asp Ile Asp Val Phe Ser
155 160 165 170
tat ggc gcc ccc cga gtc gga aac agg gct ttt gca gaa ttc ctg acc 624
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185
gta cag acc ggc gga aca ctc tac cgc att acc cac acc aat gat att 672
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200
gtc cct aga ctc ccg ccg cgc gaa ttc ggt tac agc cat tct agc cca 720
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215
gaa tac tgg atc aaa tct gga acc ctt gtc ccc gtc cgg cga cga gac 768
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Arg Arg Arg Asp
220 225 230
atc gtg aag ata gaa ggc atc gat gcc acc ggc ggc aat aac cag cct 816
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro
235 240 245 250
aac att ccg tcc atc acc gcg cac cta tgg tac ttc ggg tta att ggg 864
Asn Ile Pro Ser Ile Thr Ala His Leu Trp Tyr Phe Gly Leu Ile Gly
255 260 265
aca tgt ctt tagtg 878
Thr Cys Leu
<210> 6
<211> 291
<212> PRT
<213>Artificial sequence
<220>
<223>Synthesize construct
<400> 6
Met Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu
-20 -15 -10
Ala Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe
-5 -1 1 5 10
Asn Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn
15 20 25
Arg Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Ala Asn Ala Cys Pro
30 35 40
Glu Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser
45 50 55
Gly Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys
60 65 70
Leu Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile
75 80 85 90
Gly Asn Leu Asn Phe Glu Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly
95 100 105
Cys Arg Gly His Ala Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp
110 115 120
Thr Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr
125 130 135
Arg Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val
140 145 150
Ala Gly Ala Asp Leu Arg Gly Asn Lys Tyr Asp Ile Asp Val Phe Ser
155 160 165 170
Tyr Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr
175 180 185
Val Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile
190 195 200
Val Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Ser His Ser Ser Pro
205 210 215
Glu Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Arg Arg Arg Asp
220 225 230
Ile Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro
235 240 245 250
Asn Ile Pro Ser Ile Thr Ala His Leu Trp Tyr Phe Gly Leu Ile Gly
255 260 265
Thr Cys Leu

Claims (16)

1. a kind of variant of parent lipase, the variant, which is included in, corresponds to SEQ ID NO:The E1C of 2 mature polypeptide and Substitution at N233C position, with lipase active, and with the mature polypeptide of the parent lipase have at least 60% but Sequence identity less than 100%.
2. variant as claimed in claim 1, the wherein parent lipase are selected from：
A. a kind of lipase, the lipase is the polypeptide with following amino acid sequence, the amino acid sequence：(a) with from soft The wild type lipase of hair humicola lanuginosa strain DSM 4109 has at least 90% uniformity；(b) with the wild type lipase Compare, be included in E1's or Q249Electroneutral or negatively charged amino acid are positively charged at the surface of interior three-dimensional structure Amino acid substitution；In C-terminal including peptide add (c)；And/or (d) meets following limitation：(i) in the wild type fat The position E210 of fat enzyme includes negative electrical charge amino acid；(ii) in the area of the position 90-101 corresponding to the wild type lipase Domain includes negatively charged amino acid；And during (iii) includes at the position corresponding to the N94 of the wild type lipase Property or negative electrical charge amino acid and/or there is negative net charge in the region corresponding to the position 90-101 of the wild type lipase Or neutral net charge；
B. a kind of lipase with lipase active, the lipase and SEQ ID NO:2 have at least 60% but less than 100% Sequence identity, and corresponding to SEQ ID NO:2 T231R+N233R and D96E, D111A, D254S, G163K, Include substitution at least one or more (for example, several) position in P256T, G91T, D27R and G38A；
C. a kind of lipase, the lipase has SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 amino acid sequence Row；Or include SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 mature polypeptide is made from it.
3. the variant as any one of claim 1-2, the variant is selected from the group, the group is made up of the following：
A. a kind of polypeptide, the polypeptide and SEQ ID NO:2 mature polypeptide have at least 65%, at least 70%, at least 75%, extremely Lack 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% but small In 100% sequence identity；
B. a kind of polypeptide, the polypeptide by a kind of polynucleotide encoding, the polynucleotides low stringency condition, middle stringent condition, in- High stringency conditions, high stringency conditions or very under high stringency conditions with (i) SEQ ID NO:1 mature polypeptide encoded sequence or (ii) the total length complement of (i) is hybridized；
C. a kind of polypeptide, the polypeptide is by a kind of polynucleotide encoding, the polynucleotides and SEQ ID NO:1 mature polypeptide encoded Sequence have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%th, at least 96%, at least 97%, at least 98% or at least 99% or 100% uniformity；And
d.SEQ ID NO:One fragment of 2 mature polypeptide, the fragment has lipase active.
4. the variant as any one of claim 1-3, the variant is corresponding to SEQ ID NO:2 following position：2、 4、8、11、15、27、33、38、43、48、51、54、56、57、58、60、69、71、83、86、91、92、94、96、97、98、99、 101、111、123、150、152、163、176、179、187、188、189、198、199、200、210、216、220、224、225、 227、228、229、231、236、238、239、246、249、254、255、256、257、260、263、264、265、266、267、 Further comprise one or more (for example, several) substitution at 269 position.
5. the variant as described in precedent claims, the wherein substitution correspond to SEQ ID NO:2 V2K, Q4R, Q4V, N8R, N11R、Q15C、D27G、D27R、N33K、N33Q、G38A、E43C、D48C、F51V、S54T、E56K、D57G、S58A、V60K、 V60S、L69R、N71C、S83T、I86V、G91A、G91N、G91Q、N92D、N94K、N94R、D96E、D96G、D96L、D96W、 L97M、K98E、K98I、K98Q、E99K、E99N、N101D、N101S、D111A、T123V、A150G、A152G、G163K、 V176L、R179L、V187Y、V187W、Q188R、T189Y、T189W、H198S、T199R、N200R、E210K、E210Q、 S216P、Y220F、S224R、G225R、L227G、L227R、V228R、P229R、T231R、V236R、I238C、E239C、 G246C、Q249R、D254S、I255G、P256K、P256T、P256V、A257I、A257V、W260C、G263Q、L264A、 I265T、G266D、T267A、L269N、L269V。
6. the variant as any one of claim 1-5, the variant is corresponding to SEQ ID NO:One is included at 2 position Group substitution is made from it, and group substitution is selected from：
a.E1C N233C；
b.E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K N233C D254S P256T；
c.E1C V2K D27G N33K G38A F51V D96E D111A G163K N233C D254S P256T；
d.E1C V2K D27R N33K G38A F51V D96E D111A G163K Q188R N233C D254S P256T；
e.E1C D27R G38A G91A N92D D96L K98Q D111A G163K N233C D254S P256T；
f.E1C D27R G38A G91N N94R D96E D111A G163K S216P L227G N233C D254S P256T；
g.E1C T231R N233C；
h.E1C T231R N233C Q249R D254S；
i.E1C G225R T231R N233C；
j.E1C Q15C E43C T231R N233C；
k.E1C L227R T231R N233C；
l.E1C P229R T231R N233C；
m.E1C L227G T231R N233C；
n.E1C E99N N101S T231R N233C；
o.E1C L227G T231R N233C D254S；
p.E1C E210K L227G T231R N233C；
q.E1C D27R N33K G38A F51V D96E K98E N101D D111A G163K H198S E210K Y220F T231R N233C D254S P256T；
r.E1C D27R N33K G38A F51V S54T E56K D57G L69R D96E K98I D111A A152G G163K T231R N233C D254S P256T；
s.E1C V187Y T189Y L227G T231R N233C；
t.E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；
u.E1C V60K I86V A150G E210K L227G T231R N233C P256K；
v.E1C V187W T189W L227G T231R N233C；
w.E1C N94K D96L L227G T231R N233C；
x.E1C G91A N92D D96L K98Q L227G T231R N233C；
y.E1C N8R L227G T231R N233C；
z.E1C L227G V228R T231R N233C；
aa.E1C Q4R L227G T231R N233C；
bb.E1C N11R L227G T231R N233C；
cc.E1C S224R L227G T231R N233C；
dd.E1C L227G T231R N233C V236R；
ee.E1C N200R L227G T231R N233C；
ff.E1C T199R L227G T231R N233C；
gg.E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T；
hh.E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T；
ii.E1C D27R N33K G38A F51V D96E K98I D111A G163K H198S Y220F T231R N233C D254S P256T；
jj.E1C D27R N33K G38A F51V E56K L69R D96E K98E D111A G163K R179L T231R N233C D254S P256T A257I；
kk.E1C V2K D27R N33K G38A F51V D96E D111A G163K T231R N233C D254S P256T A257I；
ll.E1C D27R N33K G38A F51V S54T E56K D96E K98I D111A G163K T231R N233C D254S P256T A257I；
mm.E1C D27R N33K G38A F51V S54T E56K D57G D96E K98I D111A G163K T231R N233C D254S I255G P256T A257V L269V；
nn.E1C V2K D27R N33K G38A F51V L69R D96E K98E D111A G163K V176L E210K L227G T231R N233C D254S P256T；
oo.E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；
pp.E1C D27R N33K G38A F51V D96E K98E N101D D111A T123V G163K H198S E210K Y220F T231R N233C D254S P256T；
qq.E1C D27R G38A F51V L69R D96E K98E D111A G163K E210K T231R N233C D254S P256T；And
rr.E1C N11R D27R N33K D48C F51V L69R N71C E87Q K98E N101R T143A E210K G225R L227G P229R T231R N233C Q249R P250R D254S I255G P256K。
7. the variant as any one of claim 1-6, the variant has improved stabilization relative to the parent lipase Property.
8. variant as claimed in claim 7, the wherein stability are selected from the group, the group is made up of the following：Heat endurance, Stability in the presence of proteolytic enzyme, stability in the presence of surfactants, stabilization in detergent compositions Property, the stability under condition of storage.
9. a kind of composition, said composition includes the variant as any one of claim 1-8.
10. a kind of method for manufacturing composition, this method includes adding the variant as any one of claim 1-8 It is added to the step in said composition.
11. a kind of polynucleotides, variant of the polynucleotide encoding as any one of claim 1-8.
12. a kind of nucleic acid construct, the nucleic acid construct includes polynucleotides as claimed in claim 11.
13. a kind of expression vector, the expression vector includes polynucleotides as claimed in claim 11.
14. a kind of host cell, the host cell comprising polynucleotides as claimed in claim 11, as claimed in claim 12 Nucleic acid construct or expression vector as claimed in claim 13.
15. a kind of method for producing lipase Variant, this method includes：(a) cultivated under conditions of variant expression is suitable for Host cell as claimed in claim 14；And reclaim the variant (b).
16. a kind of method for obtaining lipase Variant, this method is included in corresponding to SEQ ID NO:2 E1C and N233C Position at substitution is introduced into parent lipase, the wherein variant has lipase active；And reclaim the variant.
CN201580069357.8A 2014-12-22 2015-12-18 Detergent compositions, lipase variants, and polynucleotides encoding same Active CN107109382B (en)

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